Peptides and methods for the detection of Lyme disease antibodies

ABSTRACT

The invention provides compositions (e.g., peptide compositions) useful for the detection of antibodies that bind to Borrelia antigens. The peptide compositions comprise polypeptide sequences comprising variants in the IR6 domain of the Borrelia VlsE protein. The invention also provides devices, methods, and kits comprising such peptide compositions and useful for the detection of antibodies that bind to Borrelia antigens and the diagnosis of Lyme disease.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/684,635, filed Aug. 23, 2017, which is a divisional of U.S. patentapplication Ser. No. 14/924,358, filed Oct. 27, 2015 (now U.S. Pat. No.9,766,238, issued Sep. 19, 2017), which is a continuation of U.S. patentapplication Ser. No. 14/304,809, filed Jun. 13, 2014 (now U.S. Pat. No.9,201,071, issued Dec. 1, 2015), which is a continuation of U.S. patentapplication Ser. No. 13/667,909, filed Nov. 2, 2012 (now U.S. Pat. No.8,758,772, issued Jun. 24, 2014), which claims the benefit of priorityof U.S. Provisional Application No. 61/556,061, filed Nov. 4, 2011; allof which are incorporated herein by reference in their entireties.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

The contents of the text file submitted electronically herewith areincorporated herein by reference in their entirety: A computer readableformat copy of the Sequence Listing (filename:ABAX_039_05US_SeqList_ST25.txt, date recorded: Sep. 7, 2018, file size45 kilobytes).

BACKGROUND OF THE INVENTION

Lyme disease is a debilitating condition that has become a significantpublic health concern. The disease is caused by infection with apathogenic Borrelia bacterium (a spirochete) and is transmitted by thebite of various species of Borrelia -infected Ixodes ticks. Accurate andearly detection of Lyme disease is critical to effective treatment. Theonly clinical symptom sufficient for diagnosis of Lyme disease is thepresence of erythema migrans, a rash having a distinctive bulls-eyeappearance. However, erythema migrans is only present early duringinfection and, even then, does not appear in all infected individuals.Other clinical symptoms that have been associated with Lyme disease,such as Bell's palsy, are not specific enough, either alone or incombination, to determine clinical diagnosis in the absence of erythemamigrans.

In the absence of erythema migrans, the basis for diagnosis of Lymedisease is an antibody response to a pathogenic Borrelia species, suchas Borrelia burgdorferi, Borrelia afzelli, or Borrelia garinii. In NorthAmerica, the Center for Disease Control (CDC) recommends a two-tierapproach for serodiagnosis of Lyme disease consisting of a sensitivefirst-tier assay, such as ELISA, followed by a western blot if the firsttier assay is positive or equivocal. First tier assays havetraditionally made use of a whole-cell Borrelia burgdorferi antigen orrecombinant Borrelia proteins. Such assays can be difficult tointerpret, though, and are complicated by Borrelia antibodies arisingfrom vaccination rather than infection. In addition, whole cellsonicates used in some Borrelia assays react with Treponema antibodies.

More recently, the C6 peptide assay, based on the conserved IR6 domainof the variable surface antigen (VlsE) of Borrelia, has become widelyaccepted as a first-tier assay having a high degree of sensitivity fordisseminated and late Lyme disease. The C6 peptide assay uses a single25 amino acid sequence of the Borrelia burgdorferi VlsE protein as thetest antigen. Although it has been suggested that the C6 peptide assaymay be suitable for a single-tier approach to diagnosis of Lyme disease,it is becoming apparent that the C6 assay is not sufficiently sensitivefor such purposes because it fails to detect certain strains ofinfectious Borrelia.

Accordingly, there remains a need in the art for additional assays fordetecting Borrelia antigens and serodiagnosis of Lyme disease.

SUMMARY OF THE INVENTION

The present invention is based, in part, on the discovery that certainsequence variants in the IR6 domain of the Borrelia VlsE protein providefor robust detection of an antibody response against a wide range ofBorrelia species. Accordingly, the invention provides compositions,devices, methods, and kits useful for the detection of antibodies thatbind to Borrelia antigens and the diagnosis of Lyme disease.

In one aspect, the invention provides peptides capable of binding toantibodies that recognize Borrelia antigens. In certain embodiments, thepeptides comprise a VlsE IR6 domain and a sequence from at least one(e.g., two, three, etc.) other Borrelia antigen. In certain embodiments,the at least one other Borrelia antigen is a surface antigen (e.g.,OspC, p41, or a combination thereof).

In certain embodiments, peptides of the invention comprise a sequence ofSEQ ID NO: 1,L-K-K-D-D-N-I-A-A-A-X₁₁-V-L-R-G-X₁₆-X₁₇-K-D-G-X₂₁-F-A-X₂₄-X₂₅ (SEQ IDNO: 1) wherein Xii is an amino acid selected from the group consistingof V and L, X₁₆ is an amino acid selected from the group consisting of Land I, X₁₇ is an amino acid selected from the group consisting of A andV, X₂₁ is an amino acid selected from the group consisting of R, D andN, X₂₄ is an amino acid selected from the group consisting of I, W, andY, and X₂₅ is an amino acid selected from the group consisting of K andR.

In certain embodiments, peptides of the invention comprise a sequence ofSEQ ID NO: 1 and further comprise an additional N-terminal peptidesequence. The additional N-terminal peptide sequence can comprise 1, 2,3, 4, 5, 6, 7, 8, 9, 10, or more amino acids and can be either a nativeor non-native sequence. In certain embodiments, peptides of theinvention comprise a sequence defined by SEQ ID NO: 1 and furthercomprise an additional C-terminal sequence. The additional C-terminalpeptide sequence can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or moreamino acids and can be either a native or non-native sequence. Incertain embodiments, the non-native sequence comprises a non-VlsEBorrelia antigen (e.g., a Borrelia OspC or p41 antigen).

In other embodiments, the peptides of the invention comprise a sequenceof SEQ ID NO: 2,n₁-n₂-S-P-n₅-n6-P-L-K-K-D-D-N-I-A-A-A-X₁₈-V-L-R-G-X₂₃-X₂₄-K-D-G-X₂₈-F-A-X₃₁-X₃₂-A-V-c₃₅-E-G-c₃₈-Q-Q-E-G-A-Q--P-S-C (SEQ ID NO: 2) wherein n₁ is an amino acid selected from thegroup consisting of A and V, n₂ is an amino acid selected from the groupconsisting of E and D, n₅ is an amino acid selected from the groupconsisting of K and R, n₆ is an amino acid selected from the groupconsisting of K and R, X₁₈ is an amino acid selected from the groupconsisting of V and L, X₂₃ is an amino acid selected from the groupconsisting of L and I, X₂₄ is an amino acid selected from the groupconsisting of A and V, X₂₈ is an amino acid selected from the groupconsisting of R, D and N, X₃₁ is an amino acid selected from the groupconsisting of I, W, and Y, X₃₂ is an amino acid selected from the groupconsisting of K, and R, c₃₅ is an amino acid selected from the groupconsisting of Q and R, and c₃₈ is an amino acid selected from the groupconsisting of V and A.

In certain embodiments, peptides of the invention comprise at least 25,30, 35, 40, 45, or more amino acids. In certain embodiments, peptides ofthe invention are isolated (e.g., synthetic and/or purified) peptides.In certain embodiments, peptides of the invention are conjugated to aligand. For example, in certain embodiments, the peptides arebiotinylated. In other embodiments, the peptides are conjugated toavidin, streptavidin, or neutravidin. In other embodiments, the peptidesare conjugated to a carrier protein (e.g., serum albumin, keyhole limpethemocyanin (KLH), or an immunoglobulin Fc domain). In still otherembodiments, the peptides are conjugated to a dendrimer and/or part of amultiple antigenic peptides system (MAPS).

In certain embodiments, peptides of the invention are attached to orimmobilized on a solid support. In certain embodiments, the solidsupport is a bead (e.g., a colloidal particle, nanoparticle, latex bead,etc.), a flow path in a lateral flow immunoassay device (e.g., a porousmembrane), a flow path in an analytical rotor, or a tube or well (e.g.,in a plate suitable for an ELISA assay).

In another aspect, the invention provides compositions comprising one ormore peptides of the invention. For example, in certain embodiments, theinvention provides a composition comprising a peptide comprising asequence of SEQ ID NO: 1, a peptide comprising a sequence of SEQ ID NO:2, or mixtures thereof. In certain embodiments, the compositioncomprises a mixture of two, three, four, or more different peptides ofthe invention, wherein each peptide comprises a sequence of SEQ ID NO: 1or SEQ ID NO: 2.

In certain embodiments, the peptides are conjugated to a ligand or asignaling moiety. For example, in certain embodiments, the peptides arebiotinylated. In other embodiments, the peptides are conjugated toavidin, streptavidin, or neutravidin. In other embodiments, the peptidesare conjugated to a carrier protein (e.g., serum albumin, keyhole limpethemocyanin (KLH), or an immunoglobulin Fc domain). In still otherembodiments, the peptides are conjugated to a dendrimer and/or are partof a multiple antigenic peptides system (MAPS).

In another aspect, the invention provides nucleic acids comprising asequence encoding a peptide of the invention. In addition, the inventionprovides vectors comprising such nucleic acids, and host cellscomprising such vectors. In certain embodiments, the vector is a shuttlevector. In other embodiments, the vector is an expression vector (e.g.,a bacterial or eukaryotic expression vector). In certain embodiments,the host cell is a bacterial cell. In other embodiments, the host cellis a eukaryotic cell.

In another aspect, the invention provides devices. In certainembodiments, the devices are useful for performing an immunoassay. Forexample, in certain embodiments, the device is a lateral flowimmunoassay device. In other embodiments, the device is an analyticalrotor. In other embodiments, the device is a tube or a well, e.g., in aplate suitable for an ELISA assay. In still other embodiments, thedevice is an electrochemical, optical, or opto-electronic sensor.

In certain embodiments, the device comprises a peptide of the invention.In other embodiments, the device comprises a mixture of differentpeptides of the invention. For example, in certain embodiments, thedevice comprises two, three, four, or more different peptides of theinvention. In certain embodiments, the peptide or each peptide in themixture comprises a sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In certainembodiments, the peptides are attached to or immobilized upon thedevice.

In another aspect, the invention provides methods of detecting in asample an antibody to an epitope of a Borrelia antigen. In certainembodiments, the methods comprise contacting a sample with a peptide ofthe invention, and detecting formation of an antibody-peptide complexcomprising said peptide, wherein formation of said complex is indicativeof the presence of an antibody to an epitope of a Borrelia antigen insaid sample. In certain embodiments, the Borrelia antigen is from aninfectious Borrelia species, such as Borrelia burgdorferi, Borreliaafzelli, or Borrelia garinii. In certain embodiments, the methodscomprise contacting the sample with a mixture of two, three, four, ormore different peptides of the invention.

In certain embodiments, the peptide or each peptide in the mixture is anisolated (e.g., synthetic and/or purified) peptide. In certainembodiments, the peptide or mixture of peptides is attached to orimmobilized upon a solid support. In certain embodiments, the solidsupport is a bead (e.g., a colloidal particle, nanoparticle, latex bead,etc.), a flow path in a lateral flow immunoassay device (e.g., a porousmembrane), a flow path in an analytical rotor, or a tube or a well(e.g., in a plate suitable for an ELISA assay). In certain embodiments,the solid support comprises metal, glass, a cellulose-based material(e.g., nitrocellulose), or a polymer (e.g., polystyrene, polyethylene,polypropylene, polyester, nylon, polysulfone, etc.). In certainembodiments, the peptide or mixture of different peptides is attached toa dendrimer and/or incorporated into a MAPS system. In certain otherembodiments, the peptide or mixture of different peptides is attached toBSA.

In certain embodiments, the detecting step comprises performing an ELISAassay. In other embodiments, the detecting step comprises performing alateral flow immunoassay. In other embodiments, the detecting stepcomprises performing an agglutination assay. In other embodiments, thedetecting step comprises spinning the sample in an analytical rotor. Inother embodiments, the detecting step comprises analyzing the sampleusing a Western blot, a slot blot, or a dot blot. In still otherembodiments, the detecting step comprises analyzing the sample with anelectrochemical sensor, an optical sensor, or an opto-electronic sensor.

In certain embodiments, the sample is a bodily fluid, such as blood,serum, plasma, cerebral spinal fluid, urine, mucus, or saliva. In otherembodiments, the sample is a tissue (e.g., a tissue homogenate) or acell lysate. In certain embodiments, the sample is from a wild animal(e.g., a deer or rodent, such as a mouse, chipmunk, squirrel, etc.). Inother embodiments, the sample is from a lab animal (e.g., a mouse, rat,guinea pig, rabbit, monkey, primate, etc.). In other embodiments, thesample is from a domesticated or feral animal (e.g., a dog, a cat, ahorse). In still other embodiments, the sample is from a human.

In another aspect, the invention provides methods of diagnosing Lymedisease in a subject. In certain embodiments, the methods comprisecontacting a sample from the subject with a peptide of the invention,and detecting formation of an antibody-peptide complex comprising saidpeptide, wherein formation of said complex is indicative of the subjecthaving Lyme disease. In certain embodiments, the methods comprisecontacting the sample with a mixture of two, three, four, or moredifferent peptides of the invention.

In certain embodiments, the peptide or each peptide in the mixture is anisolated (e.g., synthetic and/or purified) peptide. In certainembodiments, the peptide or mixture of different peptides is attached toor immobilized upon a solid support. For example, in certainembodiments, the solid support is a bead (e.g., a colloidal particle,nanoparticle, latex bead, etc.), a flow path in a lateral flowimmunoassay device (e.g., a porous membrane), a flow path in ananalytical rotor, or a tube or a well (e.g., in a plate suitable for anELISA assay). In certain embodiments, the solid support comprises metal,glass, a cellulose-based material (e.g., nitrocellulose), or a polymer(e.g., polystyrene, polyethylene, polypropylene, polyester, nylon,polysulfone, etc.). In certain embodiments, the peptide or mixture ofdifferent peptides is attached to a dendrimer and/or incorporated into aMAPS system. In certain other embodiments, the peptide or mixture ofdifferent peptides is attached to BSA.

In certain embodiments, the detecting step comprises performing an ELISAassay. In other embodiments, the detecting step comprises performing alateral flow immunoassay. In other embodiments, the detecting stepcomprises performing an agglutination assay. In other embodiments, thedetecting step comprises spinning the sample in an analytical rotor. Inother embodiments, the detecting step comprises analyzing the sampleusing a Western blot, a slot blot, or a dot blot. In still otherembodiments, the detecting step comprises analyzing the sample with anelectrochemical sensor, optical sensor, or opto-electronic sensor.

In certain embodiments, the sample is a bodily fluid, such as blood,serum, plasma, cerebral spinal fluid, urine, or saliva. In otherembodiments, the sample is a tissue (e.g., a tissue homogenate) or acell lysate. In certain embodiments, the subject is a wild animal (e.g.,a deer or rodent, such as a mouse, chipmunk, squirrel, etc.). In otherembodiments, the subject is a lab animal (e.g., a mouse, rat, guineapig, rabbit, monkey, primate, etc.). In other embodiments, the subjectis a domesticated or feral animal (e.g., a dog, a cat, a horse). Instill other embodiments, the subject is a human.

In yet another aspect, the invention provides kits. In certainembodiments, the kits comprise a peptide of the invention. In certainembodiments, the kits comprise two, three, four, or more differentpeptides of the invention. The peptides can comprise a sequence of SEQID NO: 1 or SEQ ID NO: 2. In certain embodiments, the peptides areattached to or immobilized on a solid support. For example, in certainembodiments, the solid support is a bead (e.g., a colloidal particle,nanoparticle, latex bead, etc.), a flow path in a lateral flowimmunoassay device, a flow path in an analytical rotor, or a tube or awell (e.g., in a plate). In certain embodiments, the peptide or mixtureof different peptides is attached to a dendrimer and/or incorporatedinto a MAPS system. In certain other embodiments, the peptide or mixtureof different peptides is attached to BSA.

In certain embodiments, the kits further comprise a population of beadsor a plate (e.g., a plate suitable for an ELISA assay). In otherembodiments, the kits further comprise a device, such as a lateral flowimmunoassay device, an analytical rotor, a Western blot, a dot blot, aslot blot, an electrochemical sensor, an optical sensor, or anopto-electronic sensor. In certain embodiments, the population of beads,the plate, or the device is useful for performing an immunoassay. Forexample, in certain embodiments, the population of beads, the plate, orthe device is useful for detecting formation of an antibody-peptidecomplex comprising an antibody from a sample and a peptide of theinvention. In certain embodiments, a peptide or a mixture of differentpeptides of the invention is attached to or immobilized on the beads,the plate, or the device.

In certain embodiments, the kits further comprise an instruction. Forexample, in certain embodiments, the kits comprise an instructionindicating how to use a peptide of the invention to detect an antibodyto a Borrelia antigen or to diagnose Lyme disease. In certainembodiments, the kits comprise an instruction indicating how to use apopulation of beads, a plate, or a device (e.g., comprising a peptide ora mixture of different peptides of the invention) to detect an antibodyto a Borrelia antigen or to diagnose Lyme disease.

Additional aspects and embodiments of the invention will be apparentfrom the detailed description that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram of an indirect sandwich assay which can be used todetect antibodies to Borrelia antigens. In this embodiment, anti-humanIgG/IgM or anti-dog IgG/IgM antibodies are immobilized to a suitablesubstrate (e.g., nitrocellulose membrane) at a test site. Antibodies ina test sample are bound by the immobilized antibodies. Test sampleantibodies to appropriate Borrelia antigens will then bind to peptidesof the invention. When the peptides of the invention are conjugated tobiotin, colloidal gold-labeled streptavidin can be used to detect thepresence of the peptides at the test site. It can be appreciated thatthe indirect sandwich assay can be operated in the reverse-that is thepeptides of the invention can be immobilized to a substrate to captureanti-Borrelia antibodies in a test sample and anti-human IgG/IgM oranti-dog IgG/IgM antibodies conjugated to a label (e.g. colloidal gold)can be used to detect the presence of the antibodies bound to theimmobilized peptides at the test site.

FIG. 2 is a diagram of a lateral flow immunoassay device based on theindirect sandwich assay of FIG. 1. In this embodiment of a lateral flowimmunoassay device, sample is applied at a sample loading pad and thenflows through the conjugate pad to the test membrane.Peptide-biotin-streptavidin-gold complexes are solubilized as the samplepasses through the conjugate pad and complexes between peptides of theinvention and appropriate anti-Borrelia antigen antibodies are thenformed. The test site comprises sample appropriate anti-IgG or anti-IgMantibodies, which bind to all antibodies in the sample. Protein L, forexample, can be used in place of anti-IgG or anti-IgM antibodies. Ifsufficient antibodies in the sample have bound to peptides of theinvention, a positive signal will appear at the test site. In anotherembodiment of a lateral flow immunoassay device, peptides of theinvention are immobilized at the test site (T) and sample appropriateanti-IgG or anti-IgM antibodies (e.g. anti-human or anti-canine)conjugated to a detectable label (e.g. colloidal gold particles) arepresent in the conjugate pad. Sample passing through the conjugate padsolubilizes the labeled antibodies and any anti-Borrelia antigenantibodies present in the test sample bind to the labeled antibodies andsuch antibody complexes are captured by the immobilized Borreliapeptides of the invention at the test site, thereby producing a positivesignal. In either embodiment, the device can further comprise a controlsite (C) at which binding partners that recognize the labeled peptide orlabeled antibody in the conjugate pad is immobilized.

FIG. 3 is a diagram of a double antigen sandwich assay which can be usedto detect antibodies to Borrelia antigens. In this embodiment, peptidesof the invention are immobilized to a suitable substrate (e.g.,nitrocellulose membrane, well of an ELISA plate) at a test site.Antibodies in a test sample are bound by the immobilized peptides of theinvention. Test sample antibodies to appropriate Borrelia antigens willthen bind to a second set of peptides of the invention that areconjugated to a detector molecule (e.g., colloidal gold, horse radishperoxidase (HRP), alkaline phosphatase (ALP)), which detects thepresence of the antibodies bound to the first set of peptidesimmobilized at the test site.

FIG. 4 is a diagram of a lateral flow immunoassay device which can beused to detect antibodies to Borrelia antigens. In this embodiment of alateral flow immunoassay device, peptides of the invention areimmobilized to a suitable substrate (e.g., nitrocellulose membrane) at atest site. Anti-Borrelia antibodies in a test sample are bound by theimmobilized peptides of the invention. Gold-conjugated protein A and/orgold-conjugated protein G is added to the system and binds to the Fcportion of the captured anti-Borrelia antibody, thereby producing apositive signal. In this embodiment, the device can further comprise acontrol site at which binding partners that recognize thegold-conjugated protein A and/or gold-conjugated protein G areimmobilized. Such binding partners may include, but are not limited to,anti-protein A, anti-protein G, mouse IgG, and/or other similar IgGmolecules.

FIG. 5 is a diagram of a lateral flow immunoassay device which can beused to detect antibodies to Borrelia antigens. In this embodiment of alateral flow immunoassay device, peptides of the invention areimmobilized to a suitable substrate (e.g., nitrocellulose membrane) at atest site. Anti-Borrelia antibodies in a test sample are bound by theimmobilized peptides of the invention. Anti-Borrelia antibodies can bedetected by gold-conjugated peptides of the invention, thereby producinga positive signal. Gold-conjugated protein A and/or gold-conjugatedprotein G can be added to the system to enhance the signal by binding tothe Fc protion of the captured anti-Borrelia antibody. In thisembodiment, the device can further comprise a control site at whichbinding partners that recognize the gold-conjugated protein A and/orgold-conjugated protein G are immobilized. Such binding partners mayinclude, but are not limited to, anti-protein A, anti-protein G, mouseIgG, and/or other similar IgG molecules.

DETAILED DESCRIPTION

As used herein, the following terms shall have the following meanings:

The term “antigen,” as used herein, refers to a molecule capable ofbeing recognized by an antibody. An antigen can be, for example, apeptide or a modified form thereof. An antigen can comprise one or moreepitopes.

The term “epitope,” as used herein, is a portion of an antigen that isspecifically recognized by an antibody. An epitope, for example, cancomprise or consist of a portion of a peptide (e.g., a peptide of theinvention). An epitope can be a linear epitope, sequential epitope, or aconformational epitope. In certain embodiments, epitopes may comprisenon-contiguous regions.

The terms “nucleic acid,” “oligonucleotide” and “polynucleotide” areused interchangeably herein and encompass DNA, RNA, cDNA, whether singlestranded or double stranded, as well as chemical modifications thereof

Single letter amino acid abbreviations used herein have their standardmeaning in the art, and all peptide sequences described herein arewritten according to convention, with the N-terminal end to the left andthe C-terminal end to the right.

Additional terms shall be defined, as required, in the detaileddescription that follows.

Compositions and Devices

The present invention is based, in part, on the discovery that certainsequence variants in the IR6 domain of the Borrelia VlsE protein providefor robust detection of an antibody response against a wide range ofBorrelia species. Accordingly, in one aspect, the invention providespeptides capable of binding to antibodies that recognize Borreliaantigens.

In certain embodiments, peptides of the invention comprise a VlsE IR6domain, or a fragment thereof, and a sequence (e.g., a sequencecomprising an epitope) from at least one (e.g., two, three, etc.) otherBorrelia antigen. In certain embodiments, the at least one otherBorrelia antigen is a surface antigen or an antigen selected from thegroup consisting of OspA, OspB, OspC, p41, and combinations thereof.Thus, for example, in certain embodiments, peptides of the inventioncomprise (i) a VlsE IR6 domain, or a fragment thereof, and (ii) asequence comprising an epitope of an OspA protein, a sequence comprisingan epitope of an OspB protein, a sequence comprising an epitope of anOspC protein, a sequence comprising an epitope of a p41 protein, or acombination of such sequences. In other embodiments, peptides of theinvention comprise (i) a VlsE IR6 domain, or a fragment thereof, (ii) asequence comprising an epitope of an OspC protein, and (iii) a sequencecomprising an epitope of a p41 protein.

In certain embodiments, peptides of the invention comprise or consist ofa sequence of SEQ ID NO: 1,L-K-K-D-D-N-I-A-A-A-X₁₁-V-L-R-G-X₁₆-X₁₇-K-D-G-X₂₁-F-A-X₂₄-X₂₅ (SEQ IDNO: 1) wherein X₁₁ is an amino acid selected from the group consistingof V and L, X₁₆ is an amino acid selected from the group consisting of Land I, X₁₇ is an amino acid selected from the group consisting of A andV, X₂₁ is an amino acid selected from the group consisting of R, D andN, X₂₄ is an amino acid selected from the group consisting of I, W, andY, and X₂₅ is an amino acid selected from the group consisting of K andR.

In certain embodiments, a peptide of the invention comprises or consistsof the sequence L-K-K-D-D-N-I-A-A-A-V-V-L-R-G-L-A-K-D-G-R-F-A-I-K (SEQID NO: 3); L-K-K-D-D-N-I-A-A-A-L-V-L-R-G-L-A-K-D-G-R-F-A-I-K (SEQ ID NO:4); L-K-K-D-D-N-I-A-A-A-V-V-L-R-G-I-A-K-D-G-R-F-A-I-K (SEQ ID NO: 5);L-K-K-D-D-N-I-A-A-A-L-V-L-R-G-I-A-K-D-G-R-F-A-I-K (SEQ ID NO: 6);L-K-K-D-D-N-I-A-A-A-V-V-L-R-G-L-V-K-D-G-R-F-A-I-K (SEQ ID NO: 7);L-K-K-D-D-N-I-A-A-A-L-V-L-R-G-L-V-K-D-G-R-F-A-I-K (SEQ ID NO: 8);L-K-K-D-D-N-I-A-A-A-V-V-L-R-G-I-V-K-D-G-R-F-A-I-K (SEQ ID NO: 9);L-K-K-D-D-N-I-A-A-A-L-V-L-R-G-I-V-K-D-G-R-F-A-I-K (SEQ ID NO: 10);L-K-K-D-D-N-I-A-A-A-V-V-L-R-G-L-A-K-D-G-D-F-A-I-K (SEQ ID NO: 11);L-K-K-D-D-N-I-A-A-A-L-V-L-R-G-L-A-K-D-G-D-F-A-I-K (SEQ ID NO: 12);L-K-K-D-D-N-I-A-A-A-V-V-L-R-G-I-A-K-D-G-D-F-A-I-K (SEQ ID NO: 13);L-K-K-D-D-N-I-A-A-A-L-V-L-R-G-I-A-K-D-G-D-F-A-I-K (SEQ ID NO: 14);L-K-K-D-D-N-I-A-A-A-V-V-L-R-G-L-V-K-D-G-D-F-A-I-K (SEQ ID NO: 15);L-K-K-D-D-N-I-A-A-A-L-V-L-R-G-L-V-K-D-G-D-F-A-I-K (SEQ ID NO: 16);L-K-K-D-D-N-I-A-A-A-V-V-L-R-G-I-V-K-D-G-D-F-A-I-K (SEQ ID NO: 17);L-K-K-D-D-N-I-A-A-A-L-V-L-R-G-I-V-K-D-G-D-F-A-I-K (SEQ ID NO: 18);L-K-K-D-D-N-I-A-A-A-V-V-L-R-G-L-A-K-D-G-N-F-A-I-K (SEQ ID NO: 19);L-K-K-D-D-N-I-A-A-A-L-V-L-R-G-L-A-K-D-G-N-F-A-I-K (SEQ ID NO: 20);L-K-K-D-D-N-I-A-A-A-V-V-L-R-G-I-A-K-D-G-N-F-A-I-K (SEQ ID NO: 21);L-K-K-D-D-N-I-A-A-A-L-V-L-R-G-I-A-K-D-G-N-F-A-I-K (SEQ ID NO: 22);L-K-K-D-D-N-I-A-A-A-V-V-L-R-G-L-V-K-D-G-N-F-A-I-K (SEQ ID NO: 23);L-K-K-D-D-N-I-A-A-A-L-V-L-R-G-L-V-K-D-G-N-F-A-I-K (SEQ ID NO: 24);L-K-K-D-D-N-I-A-A-A-V-V-L-R-G-I-V-K-D-G-N-F-A-I-K (SEQ ID NO: 25);L-K-K-D-D-N-I-A-A-A-L-V-L-R-G-I-V-K-D-G-N-F-A-I-K (SEQ ID NO: 26);L-K-K-D-D-N-I-A-A-A-V-V-L-R-G-L-A-K-D-G-R-F-A-W-K (SEQ ID NO: 27);L-K-K-D-D-N-I-A-A-A-L-V-L-R-G-L-A-K-D-G-R-F-A-W-K (SEQ ID NO: 28);L-K-K-D-D-N-I-A-A-A-V-V-L-R-G-I-A-K-D-G-R-F-A-W-K (SEQ ID NO: 29);L-K-K-D-D-N-I-A-A-A-L-V-L-R-G-I-A-K-D-G-R-F-A-W-K (SEQ ID NO: 30);L-K-K-D-D-N-I-A-A-A-V-V-L-R-G-L-V-K-D-G-R-F-A-W-K (SEQ ID NO: 31);L-K-K-D-D-N-I-A-A-A-L-V-L-R-G-L-V-K-D-G-R-F-A-W-K (SEQ ID NO: 32);L-K-K-D-D-N-I-A-A-A-V-V-L-R-G-I-V-K-D-G-R-F-A-W-K (SEQ ID NO: 33);L-K-K-D-D-N-I-A-A-A-L-V-L-R-G-I-V-K-D-G-R-F-A-W-K (SEQ ID NO: 34);L-K-K-D-D-N-I-A-A-A-V-V-L-R-G-L-A-K-D-G-D-F-A-W-K (SEQ ID NO: 35);L-K-K-D-D-N-I-A-A-A-L-V-L-R-G-L-A-K-D-G-D-F-A-W-K (SEQ ID NO: 36);L-K-K-D-D-N-I-A-A-A-V-V-L-R-G-I-A-K-D-G-D-F-A-W-K (SEQ ID NO: 37);L-K-K-D-D-N-I-A-A-A-L-V-L-R-G-I-A-K-D-G-D-F-A-W-K (SEQ ID NO: 38);L-K-K-D-D-N-I-A-A-A-V-V-L-R-G-L-V-K-D-G-D-F-A-W-K (SEQ ID NO: 39);L-K-K-D-D-N-I-A-A-A-L-V-L-R-G-L-V-K-D-G-D-F-A-W-K (SEQ ID NO: 40);L-K-K-D-D-N-I-A-A-A-V-V-L-R-G-I-V-K-D-G-D-F-A-W-K (SEQ ID NO: 41);L-K-K-D-D-N-I-A-A-A-L-V-L-R-G-I-V-K-D-G-D-F-A-W-K (SEQ ID NO: 42);L-K-K-D-D-N-I-A-A-A-V-V-L-R-G-L-A-K-D-G-N-F-A-W-K (SEQ ID NO: 43);L-K-K-D-D-N-I-A-A-A-L-V-L-R-G-L-A-K-D-G-N-F-A-W-K (SEQ ID NO: 44);L-K-K-D-D-N-I-A-A-A-V-V-L-R-G-I-A-K-D-G-N-F-A-W-K (SEQ ID NO: 45);L-K-K-D-D-N-I-A-A-A-L-V-L-R-G-I-A-K-D-G-N-F-A-W-K (SEQ ID NO: 46);L-K-K-D-D-N-I-A-A-A-V-V-L-R-G-L-V-K-D-G-N-F-A-W-K (SEQ ID NO: 47);L-K-K-D-D-N-I-A-A-A-L-V-L-R-G-L-V-K-D-G-N-F-A-W-K (SEQ ID NO: 48);L-K-K-D-D-N-I-A-A-A-V-V-L-R-G-I-V-K-D-G-N-F-A-W-K (SEQ ID NO: 49);L-K-K-D-D-N-I-A-A-A-L-V-L-R-G-I-V-K-D-G-N-F-A-W-K (SEQ ID NO: 50);L-K-K-D-D-N-I-A-A-A-V-V-L-R-G-L-A-K-D-G-R-F-A-Y-K (SEQ ID NO: 51);L-K-K-D-D-N-I-A-A-A-L-V-L-R-G-L-A-K-D-G-R-F-A-Y-K (SEQ ID NO: 52);L-K-K-D-D-N-I-A-A-A-V-V-L-R-G-I-A-K-D-G-R-F-A-Y-K (SEQ ID NO: 53);L-K-K-D-D-N-I-A-A-A-L-V-L-R-G-I-A-K-D-G-R-F-A-Y-K (SEQ ID NO: 54);L-K-K-D-D-N-I-A-A-A-V-V-L-R-G-L-V-K-D-G-R-F-A-Y-K (SEQ ID NO: 55);L-K-K-D-D-N-I-A-A-A-L-V-L-R-G-L-V-K-D-G-R-F-A-Y-K (SEQ ID NO: 56);L-K-K-D-D-N-I-A-A-A-V-V-L-R-G-I-V-K-D-G-R-F-A-Y-K (SEQ ID NO: 57);L-K-K-D-D-N-I-A-A-A-L-V-L-R-G-I-V-K-D-G-R-F-A-Y-K (SEQ ID NO: 58);L-K-K-D-D-N-I-A-A-A-V-V-L-R-G-L-A-K-D-G-D-F-A-Y-K (SEQ ID NO: 59);L-K-K-D-D-N-I-A-A-A-L-V-L-R-G-L-A-K-D-G-D-F-A-Y-K (SEQ ID NO: 60);L-K-K-D-D-N-I-A-A-A-V-V-L-R-G-I-A-K-D-G-D-F-A-Y-K (SEQ ID NO: 61);L-K-K-D-D-N-I-A-A-A-L-V-L-R-G-I-A-K-D-G-D-F-A-Y-K (SEQ ID NO: 62);L-K-K-D-D-N-I-A-A-A-V-V-L-R-G-L-V-K-D-G-D-F-A-Y-K (SEQ ID NO: 63);L-K-K-D-D-N-I-A-A-A-L-V-L-R-G-L-V-K-D-G-D-F-A-Y-K (SEQ ID NO: 64);L-K-K-D-D-N-I-A-A-A-V-V-L-R-G-I-V-K-D-G-D-F-A-Y-K (SEQ ID NO: 65);L-K-K-D-D-N-I-A-A-A-L-V-L-R-G-I-V-K-D-G-D-F-A-Y-K (SEQ ID NO: 66);L-K-K-D-D-N-I-A-A-A-V-V-L-R-G-L-A-K-D-G-N-F-A-Y-K (SEQ ID NO: 67);L-K-K-D-D-N-I-A-A-A-L-V-L-R-G-L-A-K-D-G-N-F-A-Y-K (SEQ ID NO: 68);L-K-K-D-D-N-I-A-A-A-V-V-L-R-G-I-A-K-D-G-N-F-A-Y-K (SEQ ID NO: 69);L-K-K-D-D-N-I-A-A-A-L-V-L-R-G-I-A-K-D-G-N-F-A-Y-K (SEQ ID NO: 70);L-K-K-D-D-N-I-A-A-A-V-V-L-R-G-L-V-K-D-G-N-F-A-Y-K (SEQ ID NO: 71);L-K-K-D-D-N-I-A-A-A-L-V-L-R-G-L-V-K-D-G-N-F-A-Y-K (SEQ ID NO: 72);L-K-K-D-D-N-I-A-A-A-V-V-L-R-G-I-V-K-D-G-N-F-A-Y-K (SEQ ID NO: 73);L-K-K-D-D-N-I-A-A-A-L-V-L-R-G-I-V-K-D-G-N-F-A-Y-K (SEQ ID NO: 74);L-K-K-D-D-N-I-A-A-A-V-V-L-R-G-L-A-K-D-G-R-F-A-I-R (SEQ ID NO: 75);L-K-K-D-D-N-I-A-A-A-L-V-L-R-G-L-A-K-D-G-R-F-A-I-R (SEQ ID NO: 76);L-K-K-D-D-N-I-A-A-A-V-V-L-R-G-I-A-K-D-G-R-F-A-I-R (SEQ ID NO: 77);L-K-K-D-D-N-I-A-A-A-L-V-L-R-G-I-A-K-D-G-R-F-A-I-R (SEQ ID NO: 78);L-K-K-D-D-N-I-A-A-A-V-V-L-R-G-L-V-K-D-G-R-F-A-I-R (SEQ ID NO: 79);L-K-K-D-D-N-I-A-A-A-L-V-L-R-G-L-V-K-D-G-R-F-A-I-R (SEQ ID NO: 80);L-K-K-D-D-N-I-A-A-A-V-V-L-R-G-I-V-K-D-G-R-F-A-I-R (SEQ ID NO: 81);L-K-K-D-D-N-I-A-A-A-L-V-L-R-G-I-V-K-D-G-R-F-A-I-R (SEQ ID NO: 82);L-K-K-D-D-N-I-A-A-A-V-V-L-R-G-L-A-K-D-G-D-F-A-I-R (SEQ ID NO: 83);L-K-K-D-D-N-I-A-A-A-L-V-L-R-G-L-A-K-D-G-D-F-A-I-R (SEQ ID NO: 84);L-K-K-D-D-N-I-A-A-A-V-V-L-R-G-I-A-K-D-G-D-F-A-I-R (SEQ ID NO: 85);L-K-K-D-D-N-I-A-A-A-L-V-L-R-G-I-A-K-D-G-D-F-A-I-R (SEQ ID NO: 86);L-K-K-D-D-N-I-A-A-A-V-V-L-R-G-L-V-K-D-G-D-F-A-I-R (SEQ ID NO: 87);L-K-K-D-D-N-I-A-A-A-L-V-L-R-G-L-V-K-D-G-D-F-A-I-R (SEQ ID NO: 88);L-K-K-D-D-N-I-A-A-A-V-V-L-R-G-I-V-K-D-G-D-F-A-I-R (SEQ ID NO: 89);L-K-K-D-D-N-I-A-A-A-L-V-L-R-G-I-V-K-D-G-D-F-A-I-R (SEQ ID NO: 90);L-K-K-D-D-N-I-A-A-A-V-V-L-R-G-L-A-K-D-G-N-F-A-I-R (SEQ ID NO: 91);L-K-K-D-D-N-I-A-A-A-L-V-L-R-G-L-A-K-D-G-N-F-A-I-R (SEQ ID NO: 92);L-K-K-D-D-N-I-A-A-A-V-V-L-R-G-I-A-K-D-G-N-F-A-I-R (SEQ ID NO: 93);L-K-K-D-D-N-I-A-A-A-L-V-L-R-G-I-A-K-D-G-N-F-A-I-R (SEQ ID NO: 94);L-K-K-D-D-N-I-A-A-A-V-V-L-R-G-L-V-K-D-G-N-F-A-I-R (SEQ ID NO: 95);L-K-K-D-D-N-I-A-A-A-L-V-L-R-G-L-V-K-D-G-N-F-A-I-R (SEQ ID NO: 96);L-K-K-D-D-N-I-A-A-A-V-V-L-R-G-I-V-K-D-G-N-F-A-I-R (SEQ ID NO: 97);L-K-K-D-D-N-I-A-A-A-L-V-L-R-G-I-V-K-D-G-N-F-A-I-R (SEQ ID NO: 98);L-K-K-D-D-N-I-A-A-A-V-V-L-R-G-L-A-K-D-G-R-F-A-W-R (SEQ ID NO: 99);L-K-K-D-D-N-I-A-A-A-L-V-L-R-G-L-A-K-D-G-R-F-A-W-R (SEQ ID NO: 100);L-K-K-D-D-N-I-A-A-A-V-V-L-R-G-I-A-K-D-G-R-F-A-W-R (SEQ ID NO: 101);L-K-K-D-D-N-I-A-A-A-L-V-L-R-G-I-A-K-D-G-R-F-A-W-R (SEQ ID NO: 102);L-K-K-D-D-N-I-A-A-A-V-V-L-R-G-L-V-K-D-G-R-F-A-W-R (SEQ ID NO: 103);L-K-K-D-D-N-I-A-A-A-L-V-L-R-G-L-V-K-D-G-R-F-A-W-R (SEQ ID NO: 104);L-K-K-D-D-N-I-A-A-A-V-V-L-R-G-I-V-K-D-G-R-F-A-W-R (SEQ ID NO: 105);L-K-K-D-D-N-I-A-A-A-L-V-L-R-G-I-V-K-D-G-R-F-A-W-R (SEQ ID NO: 106);L-K-K-D-D-N-I-A-A-A-V-V-L-R-G-L-A-K-D-G-D-F-A-W-R (SEQ ID NO: 107);L-K-K-D-D-N-I-A-A-A-L-V-L-R-G-L-A-K-D-G-D-F-A-W-R (SEQ ID NO: 108);L-K-K-D-D-N-I-A-A-A-V-V-L-R-G-I-A-K-D-G-D-F-A-W-R (SEQ ID NO: 109);L-K-K-D-D-N-I-A-A-A-L-V-L-R-G-I-A-K-D-G-D-F-A-W-R (SEQ ID NO: 110);L-K-K-D-D-N-I-A-A-A-V-V-L-R-G-L-V-K-D-G-D-F-A-W-R (SEQ ID NO: 111);L-K-K-D-D-N-I-A-A-A-L-V-L-R-G-L-V-K-D-G-D-F-A-W-R (SEQ ID NO: 112);L-K-K-D-D-N-I-A-A-A-V-V-L-R-G-I-V-K-D-G-D-F-A-W-R (SEQ ID NO: 113);L-K-K-D-D-N-I-A-A-A-L-V-L-R-G-I-V-K-D-G-D-F-A-W-R (SEQ ID NO: 114);L-K-K-D-D-N-I-A-A-A-V-V-L-R-G-L-A-K-D-G-N-F-A-W-R (SEQ ID NO: 115);L-K-K-D-D-N-I-A-A-A-L-V-L-R-G-L-A-K-D-G-N-F-A-W-R (SEQ ID NO: 116);L-K-K-D-D-N-I-A-A-A-V-V-L-R-G-I-A-K-D-G-N-F-A-W-R (SEQ ID NO: 117);L-K-K-D-D-N-I-A-A-A-L-V-L-R-G-I-A-K-D-G-N-F-A-W-R (SEQ ID NO: 118);L-K-K-D-D-N-I-A-A-A-V-V-L-R-G-L-V-K-D-G-N-F-A-W-R (SEQ ID NO: 119);L-K-K-D-D-N-I-A-A-A-L-V-L-R-G-L-V-K-D-G-N-F-A-W-R (SEQ ID NO: 120);L-K-K-D-D-N-I-A-A-A-V-V-L-R-G-I-V-K-D-G-N-F-A-W-R (SEQ ID NO: 121);L-K-K-D-D-N-I-A-A-A-L-V-L-R-G-I-V-K-D-G-N-F-A-W-R (SEQ ID NO: 122);L-K-K-D-D-N-I-A-A-A-V-V-L-R-G-L-A-K-D-G-R-F-A-Y-R (SEQ ID NO: 123);L-K-K-D-D-N-I-A-A-A-L-V-L-R-G-L-A-K-D-G-R-F-A-Y-R (SEQ ID NO: 124);L-K-K-D-D-N-I-A-A-A-V-V-L-R-G-I-A-K-D-G-R-F-A-Y-R (SEQ ID NO: 125);L-K-K-D-D-N-I-A-A-A-L-V-L-R-G-I-A-K-D-G-R-F-A-Y-R (SEQ ID NO: 126);L-K-K-D-D-N-I-A-A-A-V-V-L-R-G-L-V-K-D-G-R-F-A-Y-R (SEQ ID NO: 127);L-K-K-D-D-N-I-A-A-A-L-V-L-R-G-L-V-K-D-G-R-F-A-Y-R (SEQ ID NO: 128);L-K-K-D-D-N-I-A-A-A-V-V-L-R-G-I-V-K-D-G-R-F-A-Y-R (SEQ ID NO: 129);L-K-K-D-D-N-I-A-A-A-L-V-L-R-G-I-V-K-D-G-R-F-A-Y-R (SEQ ID NO: 130);L-K-K-D-D-N-I-A-A-A-V-V-L-R-G-L-A-K-D-G-D-F-A-Y-R (SEQ ID NO: 131);L-K-K-D-D-N-I-A-A-A-L-V-L-R-G-L-A-K-D-G-D-F-A-Y-R (SEQ ID NO: 132);L-K-K-D-D-N-I-A-A-A-V-V-L-R-G-I-A-K-D-G-D-F-A-Y-R (SEQ ID NO: 133);L-K-K-D-D-N-I-A-A-A-L-V-L-R-G-I-A-K-D-G-D-F-A-Y-R (SEQ ID NO: 134);L-K-K-D-D-N-I-A-A-A-V-V-L-R-G-L-V-K-D-G-D-F-A-Y-R (SEQ ID NO: 135);L-K-K-D-D-N-I-A-A-A-L-V-L-R-G-L-V-K-D-G-D-F-A-Y-R (SEQ ID NO: 136);L-K-K-D-D-N-I-A-A-A-V-V-L-R-G-I-V-K-D-G-D-F-A-Y-R (SEQ ID NO: 137);L-K-K-D-D-N-I-A-A-A-L-V-L-R-G-I-V-K-D-G-D-F-A-Y-R (SEQ ID NO: 138);L-K-K-D-D-N-I-A-A-A-V-V-L-R-G-L-A-K-D-G-N-F-A-Y-R (SEQ ID NO: 139);L-K-K-D-D-N-I-A-A-A-L-V-L-R-G-L-A-K-D-G-N-F-A-Y-R (SEQ ID NO: 140);L-K-K-D-D-N-I-A-A-A-V-V-L-R-G-I-A-K-D-G-N-F-A-Y-R (SEQ ID NO: 141);L-K-K-D-D-N-I-A-A-A-L-V-L-R-G-I-A-K-D-G-N-F-A-Y-R (SEQ ID NO: 142);L-K-K-D-D-N-I-A-A-A-V-V-L-R-G-L-V-K-D-G-N-F-A-Y-R (SEQ ID NO: 143);L-K-K-D-D-N-I-A-A-A-L-V-L-R-G-L-V-K-D-G-N-F-A-Y-R (SEQ ID NO: 144);L-K-K-D-D-N-I-A-A-A-V-V-L-R-G-I-V-K-D-G-N-F-A-Y-R (SEQ ID NO: 145); orL-K-K-D-D-N-I-A-A-A-L-V-L-R-G-I-V-K-D-G-N-F-A-Y-R (SEQ ID NO: 146).

In certain embodiments, peptides of the invention comprise a sequence ofSEQ ID NO: 1 and an additional N-terminal peptide sequence (e.g., anN-terminal extension). The additional N-terminal peptide sequence cancomprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, ormore amino acids. In certain embodiments, the N-terminal peptidesequence has a length of about 5 to about 10, about 10 to about 15,about 15 to about 20, about 20 to about 25, about 25 to about 30, about30 to about 40, or about 40 to about 50 amino acids. The additionalN-terminal peptide sequence can be a native sequence. As used herein, a“native” sequence is a peptide sequence from a naturally-occurringBorrelia VlsE sequence, or a variant thereof. In certain embodiments,the peptide sequence is a fragment of a naturally-occurring BorreliaVlsE sequence. The peptide sequence can be, e.g., from a conserved ornon-conserved region of VlsE. The peptide sequence can comprise, e.g.,an epitope, such as an immunodominant epitope or any other epitoperecognizable by a host (e.g., human, dog, etc.) immune system. VlsEproteins and peptides thereof have been described, e.g., in U.S. Pat.Nos. 6,475,492, 6,660,274, 6,719,983, 6,740,744, and 7,887,815, andEuropean Patent Nos. 0894143, 1012181, 1171605, and 1589109, thecontents of which are incorporated herein by reference.

Variant polypeptides are at least about 80, 85, 90, 95, 98, or 99%identical to a peptide shown in SEQ ID NOs: 1-146 and are alsopolypeptides of the invention. Percent sequence identity has an artrecognized meaning and there are a number of methods to measure identitybetween two polypeptide or polynucleotide sequences. See, e.g., Lesk,Ed., Computational Molecular Biology, Oxford University Press, New York,(1988); Smith, Ed., Biocomputing: Informatics And Genome Projects,Academic Press, New York, (1993); Griffin & Griffin, Eds., ComputerAnalysis Of Sequence Data, Part I, Humana Press, N.J., (1994); vonHeinje, Sequence Analysis In Molecular Biology, Academic Press, (1987);and Gribskov & Devereux, Eds., Sequence Analysis Primer, M StocktonPress, New York, (1991). Methods for aligning polynucleotides orpolypeptides are codified in computer programs, including the GCGprogram package (Devereux et al., Nuc. Acids Res. 12:387 (1984)),BLASTP, BLASTN, FASTA (Atschul et al., J Molec. Biol. 215:403 (1990)),and Bestfit program (Wisconsin Sequence Analysis Package, Version 8 forUnix, Genetics Computer Group, University Research Park, 575 ScienceDrive, Madison, Wis. 53711) which uses the local homology algorithm ofSmith and Waterman (Adv. App. Math., 2:482-489 (1981)). For example, thecomputer program ALIGN which employs the FASTA algorithm can be used,with an affine gap search with a gap open penalty of −12 and a gapextension penalty of −2.

When using any of the sequence alignment programs to determine whether aparticular sequence is, for instance, about 95% identical to a referencesequence, the parameters are set such that the percentage of identity iscalculated over the full length of the reference polynucleotide and thatgaps in identity of up to 5% of the total number of nucleotides in thereference polynucleotide are allowed.

Variants of the peptide sequences can be readily selected by one ofskill in the art, based in part on known properties of the sequence. Forexample, a variant peptide can include amino acid substitutions (e.g.,conservative amino acid substitutions) and/or deletions (e.g., small,single amino acid deletions, or deletions encompassing 2, 3, 4, 5, 10,15, 20, or more contiguous amino acids). Thus, in certain embodiments, avariant of a native peptide sequence is one that differs from anaturally-occurring sequence by (i) one or more (e.g., 2, 3, 4, 5, 6, ormore) conservative amino acid substitutions, (ii) deletion of 1 or more(e.g., 2, 3, 4, 5, 6, or more) amino acids, or (iii) a combinationthereof. Deleted amino acids can be contiguous or non-contiguous.Conservative amino acid substitutions are those that take place within afamily of amino acids that are related in their side chains and chemicalproperties. These include, e.g., (1) acidic amino acids: aspartate,glutamate; (2) basic amino acids: lysine, arginine, histidine; (3)nonpolar amino acids: alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine, tryptophan; (4) uncharged polar amino acids:glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine;(5) aliphatic amino acids: glycine, alanine, valine, leucine,isoleucine, serine, threonine, with serine and threonine optionallygrouped separately as aliphatic-hydroxyl; (6) aromatic amino acids:phenylalanine, tyrosine, tryptophan; (7) amide amino acids: asparagine,glutamine; and (9) sulfur-containing amino acids: cysteine andmethionine. See, e.g., Biochemistry, 2nd ed., Ed. by L. Stryer, W HFreeman and Co.: 1981. Methods for confirming that variant peptides aresuitable are conventional and routine.

Variants of the peptide sequences encompass variations on previouslydefined peptide sequences. For example, a previously described peptidesequence comprising a known epitope may be lengthened or shortened, atone or both ends (e.g., by about 1-3 amino acids), and/or one, two,three, four or more amino acids may be substituted by conservative aminoacids, etc. Furthermore, if a region of a protein has been identified ascontaining an epitope of interest, an investigator can “shift” theregion of interest (e.g., by about 5 amino acids in either direction)from the endpoints of the original rough region to optimize theactivity.

In certain embodiments, the additional N-terminal peptide sequence cancomprise or consist of another IR6 domain peptide. In other embodiments,the native sequence is a VlsE sequence that is naturally adjacent to theN-terminal end of a VlsE IR6 domain.

In certain embodiments, the additional N-terminal peptide sequence is anon-native sequence. As used herein, a “non-native” sequence is anyprotein sequence, whether from a Borrelia protein or otherwise, otherthan a native VlsE peptide sequence. In certain embodiments, theadditional N-terminal peptide sequence comprises an epitope of aBorrelia antigen, such as OspA, OspB, DbpA, flagella-associated proteinsFlaA (p37) and FlaB (p41), OspC (25 kd), BBK32, BmpA (p39), p21, p39,p66 or p83. Polypeptides or peptides derived from other microorganismscan also be used.

A peptide of the invention comprising an additional N-terminal peptidesequence can be designed for diagnosing Borrelia infections early afterinfection (e.g., within one to two weeks after the onset of infection).Among the pathogenic Borrelia proteins whose expression has beenrecognized in early stages of infection (e.g., to which IgM antibodyappears early after infection) are OspC, BBK32, the flagella-associatedprotein FlaB (p41), and, to a lesser extent, BmpA (p39), and theflagella-associated protein FlaA (p37). Polypeptides or peptides whichderive from those polypeptides are suitable for assays for earlyinfection. For example, some suitable linear epitopes which can be usedfor the diagnosis of early infection include peptides in OspC:PVVAESPKKP (SEQ ID NO: 147), ILMTLFLFISCNNS (SEQ ID NO: 148), and one ormore epitopes contained between amino acids 161 and 210, as reported byJobe et al. (2003) Clin Diagn Lab Immunol 10, 573-8), the contents ofwhich are incorporated herein by reference. The OspC peptides describedin U.S. Pat. No. 6,716,574, the contents of which are incorporatedherein by reference, can also be used. Other suitable regions, whichhave been shown not to contain major cross-reactive epitopes, have beenidentified in FlaB (p41), such as residues 120 to 235. See, e.g.,Crother et al. ((2003) Infect. Immun. 71, 3419-3428; Wang et al. (1999))Clin Microbial Rev 12, 633-653; and U.S. Pat. Nos. 5,618,533, 5,643,733,5,643,751, 5,932,220, and 6,617,441, the contents of each of which isincorporated herein by reference. Other peptides bearing either linearor conformational epitopes are known in the art. Methods for identifyingadditional non-native epitope sequences, particularly from variableregions of, e.g., OspC, BBK32 or DbpA, are discussed in, e.g., U.S. Pat.No. 7,887,815.

In certain embodiments, the additional N-terminal peptide sequence isfrom OspC. For example, in certain embodiments, the additionalN-terminal peptide sequence is a sequence of SEQ ID NO: 149,n₁-n₂-S-P-n₅-n₆-P (SEQ ID NO: 149) or a fragment thereof (e.g., aC-terminal fragment), wherein n₁ is an amino acid selected from thegroup consisting of A and V, n₂ is an amino acid selected from the groupconsisting of E and D, n₅ is an amino acid selected from the groupconsisting of K and R, and n₆ is an amino acid selected from the groupconsisting of K and R.

In certain embodiments, the additional N-terminal peptide sequence is acombination of sequences. For example, the additional N-terminal peptidesequence can comprise a native, a non-native sequence, or anycombination of such sequences (e.g., two or more native sequences, twoor more non-native sequence, a native and non-native sequence, etc.).

In certain embodiments, peptides of the invention comprise a sequencedefined by SEQ ID NO: 1 and further comprise an additional C-terminalsequence. The additional C-terminal peptide sequence can comprise 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or more aminoacids. The additional C-terminal peptide sequence can be a nativesequence. For example, in certain embodiments, the additional C-terminalpeptide sequence can comprise or consist of another IR6 domain peptide.In other embodiments, the native sequence is a VlsE sequence that isnaturally adjacent to the C-terminal end of a VlsE IR6 domain.

In certain embodiments, the additional C-terminal peptide sequence is anon-native sequence. In certain embodiments, the additional C-terminalpeptide sequence comprises an epitope of a Borrelia antigen, such asOspA, OspB, DbpA, flagella-associated proteins FlaA (p37) and FlaB(p41), OspC (25 kd), BBK32, BmpA (p39), p21, p39, p66 or p83, asdiscussed above. Polypeptides or peptides derived from othermicroorganisms can also be used.

In certain embodiments, the additional C-terminal peptide sequence isfrom FlaB (p41). For example, in certain embodiments, the additionalC-terminal peptide sequence is a sequence of SEQ ID NO: 150,V-c₂-E-G-c₅-Q-Q-E-G-A-Q-Q-P-S-C (SEQ ID NO: 150) or a fragment thereof(e.g., an N-terminal fragment), wherein c₂ is an amino acid selectedfrom the group consisting of Q and R, and C5 is an amino acid selectedfrom the group consisting of V and A. In other embodiments, theadditional C-terminal peptide sequence is a sequence of SEQ ID NO: 151,A-V-c₃-E-G-c₆-Q-Q-E-G-A-Q-Q-P-S-C (SEQ ID NO: 151) or a fragment thereof(e.g., an N-terminal fragment), wherein c₃ is an amino acid selectedfrom the group consisting of Q and R, and c₆ is an amino acid selectedfrom the group consisting of V and A.

In certain embodiments, the additional C-terminal peptide sequence is acombination of sequences. For example, the additional C-terminal peptidesequence can comprise a native, a non-native sequence, or anycombination of such sequences (e.g., two or more native sequences, twoor more non-native sequence, a native and non-native sequence, etc.).

In certain embodiments, peptides of the invention comprise a sequencedefined by SEQ ID NO: 1 and further comprise an additional N-terminalpeptide sequence and an additional C-terminal peptide sequence. Theadditional N-terminal and C-terminal peptide sequences can be asdescribed above. Peptides of the invention do not consist of afull-length VlsE protein. However, in certain embodiments, peptides ofthe invention can comprise a full-length VlsE protein. In otherembodiments, peptides of the invention do not comprise a full-lengthVlsE protein.

In addition to the sequences described above, the additional N-terminaland C-terminal sequences can comprise or consist of a flexible sequence,designed to better present the peptides of the invention for detectionin an immunoassay (e.g., ELISA assay, lateral flow immunoassay,agglutination assay, etc.). Such flexible sequences can be readilyidentified by persons skilled in the art.

In certain embodiments, the peptides of the invention comprise orconsist of a sequence of SEQ ID NO: 2,n₁-n₂-S-P-n₅-n₆-P-L-K-K-D-D-N-I-A-A-A-X₁₈-V-L-R-G-X₂₃-X₂₄-K-D-G-X₂₈-F-A-X₃₁-X₃₂-A-V-c₃₅-E-G-c₃₈-Q-Q-E-G-A-Q-Q-P-S-C (SEQ ID NO: 2) wherein n₁ is an amino acid selected from the groupconsisting of A and V, n₂ is an amino acid selected from the groupconsisting of E and D, n₅ is an amino acid selected from the groupconsisting of K and R, n₆ is an amino acid selected from the groupconsisting of K and R, X₁₈ is an amino acid selected from the groupconsisting of V and L, X₂₃ is an amino acid selected from the groupconsisting of L and I, X₂₄ is an amino acid selected from the groupconsisting of A and V, X₂₈ is an amino acid selected from the groupconsisting of R, D and N, X₃₁ is an amino acid selected from the groupconsisting of I, W, and Y, X₃₂ is an amino acid selected from the groupconsisting of K, and R, c₃₅ is an amino acid selected from the groupconsisting of Q and R, and c₃₈ is an amino acid selected from the groupconsisting of V and A.

In certain embodiments, peptides of the invention comprise a sequencedefined by SEQ ID NO: 2 and further comprise an additional N-terminalpeptide sequence, an additional C-terminal peptide sequence, or acombination thereof. The additional N-terminal and C-terminal peptidesequences can be as described above.

In certain embodiments, peptides of the invention comprise or consist of25 or more (e.g., 26, 27, 28, 29, or more) amino acid residues. Incertain embodiments, peptides of the invention comprise or consist of 30or more (e.g., 31, 32, 33, 34, or more) amino acid residues. In certainembodiments, peptides of the invention comprise or consist of 35 or more(e.g., 36, 37, 38, 39, or more) amino acid residues. In certainembodiments, peptides of the invention comprise or consist of 40 or more(e.g., 41, 42, 43, 44, or more) amino acid residues. In certainembodiments, peptides of the invention comprise or consist of 45 or more(e.g., 46, 47, 48, 49, or more) amino acid residues. In certainembodiments, peptides of the invention comprise or consist of 50 or more(e.g., 51, 52, 53, 54, or more) amino acid residues. In certainembodiments, peptides of the invention comprise or consist of 55, 60,65, 70, 75, 80, 85, 90, 95, 100, or more amino acid residues.

In certain embodiments, peptides of the invention comprise an epitope ofa peptide sequence described herein. For example, in certainembodiments, peptides of the invention comprise an epitope of a sequenceselected from the group consisting of SEQ ID NO: 1-146.

In certain embodiments, peptides of the invention comprise a fragment ofa peptide sequence described herein. For example, in certainembodiments, peptides of the invention comprise a fragment of a sequenceselected from the group consisting of SEQ ID NO: 1-146. The fragment canbe, e.g., at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, or 44 amino acids in length. The fragmentcan be contiguous or can include one or more deletions (e.g., a deletionof 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid residues). Incertain embodiments, the fragment comprises a sequence set forth in U.S.Pat. Nos. 6,475,492, 6,660,274, 6,719,983, 6,740,744, or 7,887,815, orEuropean Patent Nos. 0894143, 1012181, 1171605, or 1589109. In certainembodiments, the fragment does not consist of a sequence set forth inone or more of U.S. Pat. Nos. 6,475,492, 6,660,274, 6,719,983,6,740,744, and 7,887,815, and European Patent Nos. 0894143, 1012181,1171605, and 1589109. Peptides of the invention that comprise a fragmentof a peptide sequence described herein can further comprise anadditional N-terminal peptide sequence, an additional C-terminal peptidesequence, or a combination thereof. The additional N-terminal andC-terminal peptide sequences can be as described above.

Peptides of the invention comprising an additional N-terminal orC-terminal peptide sequence can further comprise a linker connecting thepeptide (e.g., a peptide of SEQ ID NO: 1 or SEQ ID NO: 2, or a fragmentthereof) with the additional N-terminal or C-terminal peptide sequence.The linker can be, e.g., a peptide spacer. Such a spacer can consist of,for example, between about one and five (e.g., about three) amino acidresidues, preferably uncharged amino acids, e.g., aliphatic residuessuch as glycine or alanine. In one embodiment, the spacer is a tripletglycine spacer. In another embodiment, the spacer is a triplet alaninespacer. In yet another embodiment, the spacer comprises both glycine andalanine residues. Alternatively, the linker can be a chemical (i.e.,non-peptide) linker.

In certain embodiments, peptides of the invention are produced bysynthetic chemistry (i.e., a “synthetic peptide”). In other embodiments,peptides of the invention are produced biologically (i.e., by cellularmachinery, such as a ribosome). In certain embodiments, peptides of theinvention are isolated. As used herein, an “isolated” peptide is apeptide that has been produced either synthetically or biologically andthen purified, at least partially, from the chemicals and/or cellularmachinery used to produce the peptide. In certain embodiments, anisolated peptide of the invention is substantially purified. The term“substantially purified,” as used herein, refers to a molecule, such asa peptide, that is substantially free of cellular material (proteins,lipids, carbohydrates, nucleic acids, etc.), culture medium, chemicalprecursors, chemicals used in synthesis of the peptide, or combinationsthereof. A peptide that is substantially purified has less than about40%, 30%, 25%, 20%, 15%, 10%, 5%, 2%, 1% or less of the cellularmaterial, culture medium, other polypeptides, chemical precursors,and/or chemicals used in synthesis of the peptide. Accordingly, asubstantially pure molecule, such as a peptide, can be at least about60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%, by dry weight, themolecule of interest. An isolated peptide of the invention can be inwater, a buffer, or in a dry form awaiting reconstitution, e.g., as partof a kit. An isolated peptide of the present invention can be in theform of a pharmaceutically acceptable salt. Suitable acids and basesthat are capable of forming salts with the peptides of the presentinvention are well known to those of skill in the art, and includeinorganic and organic acids and bases.

In certain embodiments, peptides of the invention are affinity purified.For example, in certain embodiments, the peptides of the invention arepurified by means of their ability to bind to anti-Borrelia antibodies(e.g., antibodies to VlsE proteins and, optionally, other Borreliaantigens) by contacting such antibodies with the peptides of theinvention such that peptide-antibody complexes are able to form, washingthe peptide-antibody complexes to remove impurities, and then elutingthe peptides from the antibodies. The antibodies can be, e.g., attachedto a solid support. Methods of affinity purification are well-known androutine to those skilled in the art.

In certain embodiments, peptides of the invention are modified. Thepeptides of the invention may be modified by a variety of techniques,such as by denaturation with heat and/or a detergent (e.g., SDS).Alternatively, peptides of the invention may be modified by associationwith one or more further moieties. The association can be covalent ornon-covalent, and can be, for example, via a terminal amino acid linker,such as lysine or cysteine, a chemical coupling agent, or a peptidebond. The additional moiety can be, for example, a ligand, a ligandreceptor, a fusion partner, a detectable label, an enzyme, or asubstrate that immobilizes the peptide.

Peptides of the invention can be conjugated to a ligand, such as biotin(e.g., via a cysteine or lysine residue), a lipid molecule (e.g., via acysteine residue), or a carrier protein (e.g., serum albumin, keyholelimpet hemocyanin (KLH), immunoglobulin Fc domain via e.g., a cysteineor lysine residue). Attachment to ligands, such as biotin, can be usefulfor associating the peptide with ligand receptors, such as avidin,streptavidin, polymeric streptavidin (see e.g., US 2010/0081125 and US2010/0267166, both of which are herein incorporated by reference), orneutravidin. Avidin, streptavidin, polymeric streptavidin, neutravidin,in turn, can be linked to a signaling moiety (e.g., a moiety that can bevisualized, such as colloidal gold, a fluorescent moiety, or an enzyme(horseradish peroxidase or alkaline phosphatase) or a solid substrate(e.g., an Immobilon or nitrocellulose membrane). Alternatively, thepeptides of the invention can be fused or linked to a ligand receptor,such as avidin, streptavidin, polymeric streptavidin, or neutravidin,thereby facilitating the association of the peptides with thecorresponding ligand, such as biotin and any moiety (e.g., signalingmoiety) or solid substrate attached thereto. Examples of otherligand-receptor pairs are well-known in the art and can similarly beused. In some embodiments, the peptides of the invention can be linkedor conjugated to a signaling moiety directly.

Peptides of the invention can be fused or conjugated to a fusion partner(e.g., a peptide or other moiety). In some embodiments, a fusion partnercan facilitate purification, expression of the peptide in a host cell,detection, stabilize the peptide, connecting the peptide to a surface orother entities, etc. Examples of suitable compounds for fusion partnersinclude carrier proteins (e.g., serum albumin, immunoglobulin Fc domain,dendrimer, etc.), beta-galactosidase, glutathione-S-transferase, ahistidine tag, etc. The fusion can be achieved by means of, e.g., apeptide bond. For example, peptides of the invention and fusion partnerscan be fusion proteins and can be directly fused in-frame or cancomprise a peptide linker, as discussed above in the context ofadditional N-terminal and C-terminal peptide sequences. In certainembodiments, a mixture of peptides of the invention can be linked by adendrimer, e.g., as in a MAPS structure.

In addition, peptides of the invention may be modified to include any ofa variety of known chemical groups or molecules. Such modificationsinclude, but are not limited to, glycosylation, acetylation, acylation,ADP-ribosylation, amidation, covalent attachment to polyethylene glycol(e.g., PEGylation), covalent attachment of flavin, covalent attachmentof a heme moiety, covalent attachment of a nucleotide or nucleotidederivative, covalent attachment of a lipid or lipid derivative, covalentattachment of phosphatidylinositol, cross-linking, cyclization,disulfide bond formation, demethylation, formation of covalentcross-links, formation of cystine, formation of pyroglutamate,formylation, gamma carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,proteolytic processing, phosphorylation, prenylation, racemization,selenoylation, sulfation, ubiquitination, modifications with fattyacids, transfer-RNA mediated addition of amino acids to proteins such asarginylation, etc. Analogues of an amino acid (including unnatural aminoacids) and peptides with substituted linkages are also included.Peptides of the invention that consist of any of the sequences discussedherein may be modified by any of the discussed modifications. Suchpeptides still “consist of” the amino acids.

Modifications as set forth above are well-known to those of skill in theart and have been described in great detail in the scientificliterature. Several particularly common modifications, glycosylation,lipid attachment, sulfation, gamma-carboxylation of glutamic acidresidues, hydroxylation and ADP-ribosylation, for instance, aredescribed in many basic texts, such as Proteins-Structure and MolecularProperties, 2nd ed., T. E. Creighton, W. H. Freeman and Company, NewYork (1993). Many detailed reviews are available on this subject, suchas by Wold, F., Posttranslational Covalent Modification of Proteins, B.C. Johnson, Ed., Academic Press, New York 1-12 (1983); Seifter et al.(1990) Meth. Enzymol. 182:626-646 and Rattan et al. (1992) Ann. N.Y.Acad. Sci. 663:48-62.

In certain embodiments, peptides of the invention are attached to orimmobilized on a substrate, such as a solid or semi-solid support. Theattachment can be covalent or non-covalent, and can be facilitated by amoiety associated with the peptide that enables covalent or non-covalentbinding, such as a moiety that has a high affinity to a componentattached to the carrier, support or surface. For example, the peptidecan be associated with a ligand, such as biotin, and the componentassociated with the surface can be a corresponding ligand receptor, suchas avidin. In some embodiments, the peptide can be associated with afusion partner, e.g., bovine serum albumin (BSA), which facilitates withthe attachment of the peptide to a substrate. The peptide can beattached to or immobilized on the substrate either prior to or after theaddition of a sample containing antibody during an immunoassay.

In certain embodiments, the substrate is a bead, such as a colloidalparticle (e.g., a colloidal nanoparticle made from gold, silver,platinum, copper, metal composites, other soft metals, core-shellstructure particles, or hollow gold nanospheres) or other type ofparticle (e.g., a magnetic bead or a particle or nanoparticle comprisingsilica, latex, polystyrene, polycarbonate, polyacrylate, or PVDF). Suchparticles can comprise a label (e.g., a colorimetric, chemiluminescent,or fluorescent label) and can be useful for visualizing the location ofthe peptides during immunoassays. In certain embodiments, a terminalcysteine of a peptide of the invention is used to bind the peptidedirectly to the nanoparticles made from gold, silver, platinum, copper,metal composites, other soft metals, etc.

In certain embodiments, the substrate is a dot blot or a flow path in alateral flow immunoassay device. For example, the peptides can beattached or immobilized on a porous membrane, such as a PVDF membrane(e.g., an Immobilon™ membrane), a nitrocellulose membrane, polyethylenemembrane, nylon membrane, or a similar type of membrane.

In certain embodiments, the substrate is a flow path in an analyticalrotor. In other embodiments, the substrate is a tube or a well, such asa well in a plate (e.g., a microtiter plate) suitable for use in anELISA assay. Such substrates can comprise glass, cellulose-basedmaterials, thermoplastic polymers, such as polyethylene, polypropylene,or polyester, sintered structures composed of particulate materials(e.g., glass or various thermoplastic polymers), or cast membrane filmcomposed of nitrocellulose, nylon, polysulfone, or the like. A substratecan be sintered, fine particles of polyethylene, commonly known asporous polyethylene, for example, 0.2-15 micron porous polyethylene fromChromex Corporation (Albuquerque, N. Mex.). All of these substratematerials can be used in suitable shapes, such as films, sheets, orplates, or they may be coated onto or bonded or laminated to appropriateinert carriers, such as paper, glass, plastic films, or fabrics.Suitable methods for immobilizing peptides on solid phases includeionic, hydrophobic, covalent interactions and the like.

Accordingly, in another aspect, the invention provides devices. Incertain embodiments, the devices are useful for performing animmunoassay. For example, in certain embodiments, the device is alateral flow immunoassay device. In other embodiments, the device is ananalytical rotor. In other embodiments, the device is a dot blot, slotblot, or Western blot. In other embodiments, the device is a tube or awell, e.g., in a plate suitable for an ELISA assay. In still otherembodiments, the device is an electrochemical sensor, an optical sensor,or an opto-electronic sensor.

In certain embodiments, the device comprises a peptide of the invention.In other embodiments, the device comprises a mixture of differentpeptides of the invention. For example, in certain embodiments, thedevice comprises two, three, four, or more different peptides of theinvention. In certain embodiments, the peptide or each peptide in themixture comprises a sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In certainembodiments, the peptides are attached to or immobilized upon thedevice.

In another aspect, the invention provides compositions comprising one ormore peptides of the invention. For example, in certain embodiments, theinvention provides a composition comprising a peptide comprising asequence of SEQ ID NO: 1, a peptide comprising a sequence of SEQ ID NO:2, or mixtures thereof. In certain embodiments, the compositioncomprises a mixture of 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40,50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400, 500, or more peptides(e.g., all possible peptides defined by SEQ ID NO: 1 or SEQ ID NO: 2).In certain embodiments, the peptides are modified (e.g., by associationwith one or more further moieties), as described herein.

In certain embodiments, the compositions comprise one or more peptidesof the invention and one or more additional peptides, such as a Borreliapeptide or antigen, a peptide or antigen from one or more infectiousBorrelia species, or a peptide or antigen from one or more causativeagents of Lyme disease. The Borrelia peptide or antigen can be anyBorrelia peptide or antigen described herein (e.g., an OspA, OspB, DbpA,flagella-associated proteins FlaA (p37) and FlaB (p41), OspC (25 kd),BBK32, BmpA (p39), p21, p39, p66, p83, or VlsE protein), or any fragmentor epitope thereof. Some suitable Borrelia peptides have been described,e.g., in U.S. Pat. No. 7,887,815. The combination may comprise acocktail (a simple mixture) of individual peptides or polypeptide, itmay be in the form of a fusion peptide or polypeptide (e.g., amultimeric peptide), or the peptides may be linked by a dendrimer (e.g.,as in a MAPS structure) optionally through a linking residue (e.g.lysine residue). A peptide of the invention may be fused at itsN-terminus or C-terminus to another suitable peptide. Two or more copiesof a peptide of the invention may be joined to one another, alone or incombination with one or more additional peptides. Combinations of fusedand unfused peptides or polypeptides can be used. In one embodiment, theadditional peptide(s) contain B-cell and/or T-cell epitopes from aBorrelia peptide or antigen, a peptide or antigen from an infectiousBorrelia species, or a peptide or antigen from a causative agent of Lymedisease.

In another aspect, the invention provides nucleic acids comprising asequence encoding a peptide of the invention. Nucleic acids of theinvention contain less than an entire microbial genome and can besingle- or double-stranded. A nucleic acid can be RNA, DNA, cDNA,genomic DNA, chemically synthesized RNA or DNA or combinations thereof.The nucleic acids can be purified free of other components, such asproteins, lipids and other polynucleotides. For example, the nucleicacids can be 50%, 75%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% purified.The nucleic acids of the invention encode the peptides described herein.In certain embodiments, the nucleic acids encode a peptide having thesequence of SEQ ID NO: 1-146 or combinations thereof. Nucleic acids ofthe invention can comprise other nucleotide sequences, such as sequencescoding for linkers, signal sequences, TMR stop transfer sequences,transmembrane domains, or ligands useful in protein purification such asglutathione-S-transferase, histidine tag, and staphylococcal protein A.

Nucleic acids of the invention can be isolated. An “isolated” nucleicacid is one that is not immediately contiguous with one or both of the5′ and 3′ flanking genomic sequences that it is naturally associatedwith. An isolated nucleic acid can be, e.g., a recombinant DNA moleculeof any length, provided that the nucleic acid sequence naturally foundimmediately flanking the recombinant DNA molecule in anaturally-occurring genome is removed or absent. Isolated nucleic acidscan also include non-naturally occurring nucleic acid molecules. Nucleicacids of the invention can also comprise fragments that encodeimmunogenic peptides. Nucleic acids of the invention can encodefull-length polypeptides, peptide fragments, and variant or fusionpeptides.

Nucleic acids of the invention can be isolated, at least in part, fromnucleic acid sequences present in, for example, a biological sample,such as blood, serum, saliva, or tissue from an infected individual.Nucleic acids can also be synthesized in the laboratory, for example,using an automatic synthesizer. An amplification method such as PCR canbe used to amplify nucleic acids, at least in part, from either genomicDNA or cDNA encoding the polypeptides.

Nucleic acids of the invention can comprise coding sequences fornaturally occurring polypeptides or can encode altered sequences that donot occur in nature. If desired, nucleic acids can be cloned into anexpression vector comprising expression control elements, including forexample, origins of replication, promoters, enhancers, or otherregulatory elements that drive expression of the polynucleotides of theinvention in host cells. An expression vector can be, for example, aplasmid, such as pBR322, pUC, or ColE1, or an adenovirus vector, such asan adenovirus Type 2 vector or Type 5 vector. Optionally, other vectorscan be used, including but not limited to Sindbis virus, simian virus40, alphavirus vectors, poxvirus vectors, and cytomegalovirus andretroviral vectors, such as murine sarcoma virus, mouse mammary tumorvirus, Moloney murine leukemia virus, and Rous sarcoma virus.Minichromosomes such as MC and MC1, bacteriophages, phagemids, yeastartificial chromosomes, bacterial artificial chromosomes, virusparticles, virus-like particles, cosmids (plasmids into which phagelambda cos sites have been inserted) and replicons (genetic elementsthat are capable of replication under their own control in a cell) canalso be used.

Methods for preparing polynucleotides operably linked to an expressioncontrol sequence and expressing them in a host cell are well-known inthe art. See, e.g., U.S. Pat. No. 4,366,246. A nucleic acid of theinvention is operably linked when it is positioned adjacent to or closeto one or more expression control elements, which direct transcriptionand/or translation of the polynucleotide.

Thus, for example, a peptide of the invention can be producedrecombinantly following conventional genetic engineering techniques. Toproduce a recombinant peptide of the invention, a nucleic acid encodingthe peptide is inserted into a suitable expression system. Generally, arecombinant molecule or vector is constructed in which thepolynucleotide sequence encoding the selected peptide is operably linkedto an expression control sequence permitting expression of the peptide.Numerous types of appropriate expression vectors are known in the art,including, e.g., vectors containing bacterial, viral, yeast, fungal,insect or mammalian expression systems. Methods for obtaining and usingsuch expression vectors are well-known. For guidance in this and othermolecular biology techniques used for compositions or methods of theinvention, see, e.g., Sambrook et al., Molecular Cloning, A LaboratoryManual, current edition, Cold Spring Harbor Laboratory, New York; Milleret al, Genetic Engineering, 8:277-298 (Plenum Press, current edition),Wu et al., Methods in Gene Biotechnology (CRC Press, New York, N.Y.,current edition), Recombinant Gene Expression Protocols, in Methods inMolecular Biology, Vol. 62, (Tuan, ed., Humana Press, Totowa, N.J.,current edition), and Current Protocols in Molecular Biology, (Ausabelet al, Eds.,) John Wiley & Sons, NY (current edition), and referencescited therein.

Accordingly, the invention also provides vectors comprising nucleicacids of the invention, and host cells comprising such vectors. Incertain embodiments, the vector is a shuttle vector. In otherembodiments, the vector is an expression vector (e.g., a bacterial oreukaryotic expression vector). In certain embodiments, the host cell isa bacterial cell. In other embodiments, the host cell is a eukaryoticcell.

Suitable host cells or cell lines for the recombinant nucleic acids orvectors of the invention transfection by this method include bacterialcells. For example, various strains of E. coli (e.g., HB101, MC1061) arewell-known as host cells in the field of biotechnology. Various strainsof B. subtilis, Pseudomonas, Streptomyces, and other bacilli and thelike can also be employed in this method. Alternatively, a peptide ofthe invention can be expressed in yeast, insect, mammalian, or othercell types, using conventional procedures. Cell-free in vitro synthesisand/or enzyme-mediated synthetic machineries may also be used.

The present invention also provides a method for producing a recombinantpeptide or polypeptide, which involves transfecting or transforming,e.g., by conventional means such as electroporation, a host cell with atleast one expression vector containing a polynucleotide of the inventionunder the control of an expression control sequence (e.g., atranscriptional regulatory sequence). The transfected or transformedhost cell is then cultured under conditions that allow expression of thepeptide or polypeptide. The expressed peptide or polypeptide isrecovered, isolated, and optionally purified from the cell (or from theculture medium, if expressed extracellularly) by appropriate means knownto one of skill in the art, including liquid chromatography such asnormal or reversed phase, using HPLC, FPLC and the like, affinitychromatography, such as with inorganic ligands or monoclonal antibodies,size exclusion chromatography, immobilized metal chelate chromatography,gel electrophoresis, and the like. One of skill in the art may selectthe most appropriate isolation and purification techniques withoutdeparting from the scope of this invention. One skilled in the art candetermine the purity of the peptide or polypeptide by using standardmethods including, e.g., polyacrylamide gel electrophoresis (e.g.,SDS-PAGE), capillary electrophoresis, column chromatography (e.g., highperformance liquid chromatography (HPLC)), amino-terminal amino acidanalysis, and quantitative amino acid analysis.

Methods

In another aspect, the invention provides methods of detecting in asample an antibody to an epitope of a Borrelia antigen. In certainembodiments, the methods comprise contacting a sample with a peptide ofthe invention, and detecting formation of an antibody-peptide complexcomprising said peptide, wherein formation of said complex is indicativeof the presence of an antibody to an epitope of a Borrelia antigen insaid sample. In certain embodiments, the Borrelia antigen is from aninfectious Borrelia species. In certain embodiments, the Borreliaantigen is from a pathogenic Borrelia species, such as Borreliaburgdorferi sensu stricto, Borrelia afzelli, or Borrelia garinii. Otherspecies of Borrelia which have been implicated in Lyme disease, such asB. lusitaniae and B. valaisianae, can also be detected using the methodsof the invention, provided they induce antibodies which can reactspecifically with a peptide of the invention. Thus, it is to beunderstood that the term “pathogenic Borrelia,” as used herein, refersto any such Borrelia species that causes Lyme disease.

In certain embodiments, the methods comprise contacting the sample witha mixture of two, three, four, or more (e.g., 5, 6, 7, 8, 9, 10, 15, 20,25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400, 500, ormore) different peptides of the invention. In certain embodiments, themethods comprise contacting the sample with a mixture of one or morepeptides of the invention and one or more other peptides (e.g., aBorrelia peptide, or antigenic fragment or epitope thereof, such as anOspA, OspB, DbpA, flagella-associated proteins FlaA (p37) and FlaB(p41), OspC (25 kd), BBK32, BmpA (p39), p21, p39, p66, p83, or VlsEprotein).

In certain embodiments, the peptide or each peptide in the mixture is anisolated (e.g., synthetic and/or purified) peptide. In certainembodiments, the peptide or mixture of peptides is attached to orimmobilized upon a solid support. In certain embodiments, the solidsupport is a bead (e.g., a colloidal particle, nanoparticle, latex bead,etc.), a flow path in a lateral flow immunoassay device (e.g., a porousmembrane), a flow path in an analytical rotor, a blot (Western blot, dotblot, or slot blot), a tube or a well (e.g., in a plate suitable for anELISA assay), or a sensor (e.g., an electrochemical, optical, oropto-electronic sensor).

In certain embodiments, the detecting step comprises performing an ELISAassay. In other embodiments, the detecting step comprises performing alateral flow immunoassay. In other embodiments, the detecting stepcomprises performing an agglutination assay. In other embodiments, thedetecting step comprises spinning the sample in an analytical rotor. Instill other embodiments, the detecting step comprises analyzing thesample with an electrochemical, optical, or opto-electronic sensor.

There are a number of different conventional assays for detectingformation of an antibody-peptide complex comprising a peptide of theinvention. For example, the detecting step can comprise performing anELISA assay, performing a lateral flow immunoassay, performing anagglutination assay, performing a Western blot, slot blot, or dot blot,analyzing the sample in an analytical rotor, or analyzing the samplewith an electrochemical, optical, or opto-electronic sensor. Thesedifferent assays are described above and/or are well-known to thoseskilled in the art.

In one embodiment, the methods involve detecting the presence ofnaturally occurring antibodies against a Borrelia antigen (e.g., theantigen of a pathogenic Borrelia, such as B. Burgdorferi) which areproduced by the infected subject's immune system in its biologicalfluids or tissues, and which are capable of binding specifically to apeptide of the invention or combinations of a peptide of the inventionand, optionally, one or more suitable additional antigenic polypeptidesor peptides.

Suitable immunoassay methods typically include: receiving or obtaining(e.g., from a patient) a sample of body fluid or tissue likely tocontain antibodies; contacting (e.g., incubating or reacting) a sampleto be assayed with a peptide of the invention, under conditionseffective for the formation of a specific peptide-antibody complex(e.g., for specific binding of the peptide to the antibody); andassaying the contacted (reacted) sample for the presence of anantibody-peptide reaction (e.g., determining the amount of anantibody-peptide complex). The presence of an elevated amount of theantibody-peptide complex indicates that the subject was exposed to andinfected with an infectious species. A peptide, including a modifiedform thereof, which “binds specifically” to (e.g., “is specific for” orbinds “preferentially” to) an antibody against a Borrelia antigeninteracts with the antibody, or forms or undergoes a physicalassociation with it, in an amount and for a sufficient time to allowdetection of the antibody. By “specifically” or “preferentially,” it ismeant that the peptide has a higher affinity (e.g., a higher degree ofselectivity) for such an antibody than for other antibodies in a sample.For example, the peptide can have an affinity for the antibody of atleast about 1.5-fold, 2-fold, 2.5-fold, 3-fold, or higher than for otherantibodies in the sample. Such affinity or degree of specificity can bedetermined by a variety of routine procedures, including, e.g.,competitive binding studies. In an ELISA assay, a positive response isdefined as a value 2 or 3 standard deviations greater than the meanvalue of a group of healthy controls. In some embodiments, a second tierassay is required to provide an unequivocal serodiagnosis of Lymedisease.

Phrases such as “sample containing an antibody” or “detecting anantibody in a sample” are not meant to exclude samples or determinations(e.g., detection attempts) where no antibody is contained or detected.In a general sense, this invention involves assays to determine whetheran antibody produced in response to infection with an infectiousBorrelia is present in a sample, irrespective of whether or not it isdetected.

Conditions for reacting peptides and antibodies so that they reactspecifically are well-known to those of skill in the art. See, e.g.,Current Protocols in Immunology (Coligan et al., editors, John Wiley &Sons, Inc).

The methods comprise receiving or obtaining a sample of body fluid ortissue likely to contain antibodies from a subject. The antibodies canbe, e.g., of IgG, IgE, IgD, IgM, or IgA type. Generally, IgM and/or IgAantibodies are detected, e.g., for detection at early stages ofinfection. Although, in the case of a Borrelia infection, IgM antibodiescan persist for a long time. IgG antibodies can be detected when some ofthe additional peptides discussed above are used in the method (e.g.,peptides for the detection of flagellum proteins). The sample ispreferably easy to obtain and may be serum or plasma derived from avenous blood sample or even from a finger prick. Tissue from other bodyparts or other bodily fluids, such as cerebro-spinal fluid (CSF),saliva, gastric secretions, mucus, urine, etc., are known to containantibodies and may be used as a source of the sample.

Once the peptide antigen and sample antibody are permitted to react in asuitable medium, an assay is performed to determine the presence orabsence of an antibody-peptide reaction. Among the many types ofsuitable assays, which will be evident to a skilled worker, areimmunoprecipitation and agglutination assays.

In certain embodiments of the invention, the assay comprises:immobilizing the antibody(s) in the sample, e.g., directly or indirectlyvia binding to peptides of the invention; adding a peptide of theinvention; and detecting the degree of antibody bound to the peptide,e.g., by the peptide being labeled or by adding a labeled substance,such as a labeled binding partner (e.g., streptavidin-colloidal goldcomplex) or a labeled antibody which specifically recognizes thepeptide. See, e.g., FIG. 1. In other embodiments, the assay comprises:immobilizing a peptide of the invention; adding the sample containingantibodies; and detecting the amount of antibody bound to the peptide,e.g., by adding another peptide of the invention conjugated, directly orindirectly, to a label (e.g., colloidal gold complex, fluorescent label,enzyme (e.g., horseradish peroxidase or alkaline phosphatase)) or byadding a labeled substance, such as a binding partner or a labeledantibody which specifically recognizes the sample antibodies (e.g.,anti-human IgG antibodies, anti-human IgM antibodies, anti-dog IgGantibodies, anti-dog IgM antibodies, protein A, protein G, protein L,etc.) or combinations thereof. See, e.g., FIG. 3. In other embodiments,the assay comprises: immobilizing a peptide of the invention; adding thesample containing antibodies; and detecting the amount of antibody boundto the peptide, e.g., by adding a first binding partner whichspecifically recognizes the sample antibodies (e.g., anti-human IgGantibodies, anti-human IgM antibodies, anti-dog IgG antibodies, anti-dogIgM antibodies, protein A, protein G, protein L, etc.), and furtheradding a second binding partner (e.g., protein A, protein G, protein L,etc.), wherein the second binding partner is labeled and recognizes saidfirst binding partner. In still other embodiments, the assay comprises:reacting the peptide and the sample containing antibodies without any ofthe reactants being immobilized, and then detecting the amount ofcomplexes of antibody and peptide, e.g., by the peptide being labeled orby adding a labeled substance, such as a labeled binding partner (e.g.,streptavidin-colloidal gold complex) or a labeled antibody whichspecifically recognizes the peptide.

Immobilization of a peptide of the invention can be either covalent ornon-covalent, and the non-covalent immobilization can be non-specific(e.g., non-specific binding to a polystyrene surface in, e.g., amicrotiter well). Specific or semi-specific binding to a solid orsemi-solid carrier, support or surface, can be achieved by the peptidehaving, associated with it, a moiety which enables its covalent ornon-covalent binding to the solid or semi-solid carrier, support orsurface. For example, the moiety can have affinity to a componentattached to the carrier, support or surface. In this case, the moietymay be, e.g., a biotin or biotinyl group or an analogue thereof bound toan amino acid group of the peptide, such as 6-aminohexanoic acid, andthe component is then avidin, streptavidin, neutravidin, or an analoguethereof. An alternative is a situation in which the moiety has the aminoacid sequence His-His-His-His-His-His (SEQ ID NO: 152) and the carriercomprises a Nitrilotriacetic Acid (NTA) derivative charged with Ni⁺⁺ orCo⁺⁺ ions. In certain embodiments, the moiety is a fusion partner, e.g.,BSA. In exemplary embodiments, peptides of the invention may beconjugated to BSA via N-terminal and/or C-terminal residues of thepeptides. In one embodiment, one, two, three, four, five, 10, 15, 20,25, 30 or more peptides of the invention may be substituted into, e.g.,conjugated with BSA. As would be understood by one skilled in the art,substitution levels may impact the sensitivity of the assay. Lowerconcentrations of highly substituted BSA are needed to achievesensitivity offered by high concentrations of BSA-peptide containingfewer molecules of peptide. In certain other embodiments, the fusionpartner may be MAPS. In certain exemplary embodiments, MAPS may consistof 4, 8, or more asymmetric branches.

Suitable carriers, supports, and surfaces include, but are not limitedto, beads (e.g., magnetic beads, colloidal particles or nanoparticles,such as colloidal gold, or nanoparticles comprising silica, latex,polystyrene, polycarbonate, or PDVF), latex of co-polymers such asstyrene-divinyl benzene, hydroxylated styrene-divinyl benzene,polystyrene, carboxylated polystyrene, beads of carbon black,non-activated or polystyrene or polyvinyl chloride activated glass,epoxy-activated porous magnetic glass, gelatin or polysaccharideparticles or other protein particles, red blood cells, mono- orpolyclonal antibodies or Fab fragments of such antibodies.

The protocols for immunoassays using antigens for detection of specificantibodies are well known in art. For example, a conventional sandwichassay can be used, or a conventional competitive assay format can beused. For a discussion of some suitable types of assays, see CurrentProtocols in Immunology (supra). In certain embodiments, a peptide ofthe invention is immobilized on a solid or semi-solid surface or carrierby means of covalent or non-covalent binding, either prior to or afterthe addition of the sample containing antibody.

Devices for performing specific binding assays, especially immunoassays,are known and can be readily adapted for use in the present methods.Solid phase assays, in general, are easier to perform than heterogeneousassay methods which require a separation step, such as precipitation,centrifugation, filtration, chromatography, or magnetism, becauseseparation of reagents is faster and simpler. Solid-phase assay devicesinclude microtiter plates, flow-through assay devices (e.g., lateralflow immunoassay devices), dipsticks, and immunocapillary orimmunochromatographic immunoassay devices.

In embodiments of the invention, the solid or semi-solid surface orcarrier is the floor or wall in a microtiter well, a filter surface ormembrane (e.g., a nitrocellulose membrane or a PVDF (polyvinylidenefluoride) membrane, such as an Immobilon™ membrane), a hollow fiber, abeaded chromatographic medium (e.g., an agarose or polyacrylamide gel),a magnetic bead, a fibrous cellulose matrix, an HPLC matrix, an FPLCmatrix, a substance having molecules of such a size that the moleculeswith the peptide bound thereto, when dissolved or dispersed in a liquidphase, can be retained by means of a filter, a substance capable offorming micelles or participating in the formation of micelles allowinga liquid phase to be changed or exchanged without entraining themicelles, a water-soluble polymer, or any other suitable carrier,support or surface.

In some embodiments of the invention, the peptide is provided with asuitable label which enables detection. Conventional labels may be usedwhich are capable, alone or in concert with other compositions orcompounds, of providing a detectable signal. Suitable detection methodsinclude, e.g., detection of an agent which is tagged, directly orindirectly, with a fluorescent label by immunofluorescence microscopy,including confocal microscopy, or by flow cytometry (FACS), detection ofa radioactively labeled agent by autoradiography, electron microscopy,immunostaining, subcellular fractionation, or the like. In oneembodiment, a radioactive element (e.g., a radioactive amino acid) isincorporated directly into a peptide chain; in another embodiment, afluorescent label is associated with a peptide via biotin/avidininteraction, association with a fluorescein conjugated antibody, or thelike. In one embodiment, a detectable specific binding partner for theantibody is added to the mixture. For example, the binding partner canbe a detectable secondary antibody or other binding agent (e.g., proteinA, protein G, protein L) which binds to the first antibody. Thissecondary antibody or other binding agent can be labeled, e.g., with aradioactive, enzymatic, fluorescent, luminescent, or other detectablelabel, such as an avidin/biotin system. In another embodiment, thebinding partner is a peptide of the invention, which can be conjugateddirectly or indirectly (e.g. via biotin/avidin interaction) to anenzyme, such as horseradish peroxidase or alkaline phosphatase or othersignaling moeity. In the case of enzyme, the detectable signal isproduced by adding a substrate of the enzyme that produces a detectablesignal, such as a chromogenic, fluorogenic, or chemiluminescentsubstrate.

A “detection system” for detecting bound peptide, as used herein, maycomprise a detectable binding partner, such as an antibody specific forthe peptide. In one embodiment, the binding partner is labeled directly.In another embodiment, the binding partner is attached to a signalgenerating reagent, such as an enzyme that, in the presence of asuitable substrate, can produce a detectable signal. A surface forimmobilizing the peptide may optionally accompany the detection system.

In embodiments of the invention, the detection procedure comprisesvisibly inspecting the antibody-peptide complex for a color change, orinspecting the antibody-peptide complex for a physical-chemical change.Physical-chemical changes may occur with oxidation reactions or otherchemical reactions. They may be detected by eye, using aspectrophotometer, or the like.

A particularly useful assay format is a lateral flow immunoassay format.Antibodies to human or animal (e.g., dog, mouse, deer, etc.)immunoglobulins, or staph A or G protein antibodies, can be labeled witha signal generator or reporter (e.g., colloidal gold) that is dried andplaced on a glass fiber pad (sample application pad or conjugate pad).The diagnostic peptide is immobilized on membrane, such asnitrocellulose or a PVDF (polyvinylidene fluoride) membrane (e.g., anImmobilon™ membrane). When a solution of sample (blood, serum, etc.) isapplied to the sample application pad (or flows through the conjugatepad), it dissolves the labeled reporter, which then binds to allantibodies in the sample. The resulting complexes are then transportedinto the next membrane (PVDF or nitrocellulose containing the diagnosticpeptide) by capillary action. If antibodies against the diagnosticpeptide are present, they bind to the diagnostic peptide striped on themembrane, thereby generating a signal (e.g., a band that can be seen orvisualized). An additional antibody specific to the labeled antibody ora second labeled antibody can be used to produce a control signal.

An alternative format for the lateral flow immunoassay comprises thepeptides or compositions of the invention being conjugated to a ligand(e.g., biotin) and complexed with labeled ligand receptor (e.g.,streptavidin-colloidal gold). The labeled peptide complexes can beplaced on the sample application pad or conjugate pad. Anti-humanIgG/IgM or anti-animal (e.g., dog, mouse, deer) IgG/IgM antibodies orother peptides of the invention are immobilized on a membrane, such asnitrocellulose of PVDF, at a test site (e.g., a test line). When sampleis added to the sample application pad, antibodies in the sample reactwith the labeled peptide complexes such that antibodies that bind topeptides of the invention become indirectly labeled. The antibodies inthe sample are then transported into the next membrane (PVDF ornitrocellulose containing the diagnostic peptide) by capillary actionand bind to the immobilized anti-human IgG/IgM or anti-animal IgG/IgMantibodies (or protein A, protein G, protein L, or combinations thereof)or immobilized peptides of the invention. If any of the sampleantibodies are bound to the labeled peptides of the invention, the labelassociated with the peptides can be seen or visualized at the test site.One embodiment of this type of lateral flow device is shown in FIG. 2.Another embodiment of this type of lateral flow device in which thepeptides of the invention are used both as the immobilized capture agentat a test site and as a soluble labeled complex to react with antibodiesin a sample is shown in FIG. 3. Suitable controls for this assay caninclude, e.g., a chicken IgY-colloidal gold conjugate located at thesample application pad or conjugate pad, and an anti-chicken IgYantibody immobilized at a control site located proximal to the testsite.

In the embodiments utilizing the lateral flow immunoassay formatdescribed above, the lateral flow device may comprise two ports: asample port, which is positioned between a conjugate pad (containing alabeled analyte-binding partner) and a test site or line (containing animmobilized analyte-binding partner) and a chase port, which ispositioned upstream (e.g. toward the end of the device away from testsite) of the conjugate pad. In such devices comprising two ports, sampleis deposited upstream of the test site via the sample port and fluidflow through the conjugate pad is initiated by depositing solution (e.g.diluent, buffer, or the like) via the chase port. The “chase” solutiondissolves the labeled reagents in the conjugate pad and flows through tointeract with the same and immobilized reagents at the test site.

Another assay for the screening of blood products or other physiologicalor biological fluids is an enzyme linked immunosorbent assay, i.e., anELISA. Typically in an ELISA, isolated peptides or compositions of theinvention are adsorbed to the surface of a microtiter well directly orthrough a capture matrix (e.g., an antibody). Residual, non-specificprotein-binding sites on the surface are then blocked with anappropriate agent, such as bovine serum albumin (BSA), heat-inactivatednormal goat serum (NGS), or BLOTTO (a buffered solution of nonfat drymilk which also contains a preservative, salts, and an antifoamingagent). The well is then incubated with a biological sample suspected ofcontaining specific anti-Borrelia (e.g., B. burgdorferi) antibody. Thesample can be applied neat, or more often it can be diluted, usually ina buffered solution which contains a small amount (0.1-5.0% by weight)of protein, such as BSA, NGS, or BLOTTO. After incubating for asufficient length of time to allow specific binding to occur, the wellis washed to remove unbound protein and then incubated with an optimalconcentration of an appropriate anti-immunoglobulin antibody (e.g., forhuman subjects, an anti-human immunoglobulin (aHulg) from anotheranimal, such as dog, mouse, cow, etc.) or another peptide of theinvention that is conjugated to an enzyme or other label by standardprocedures and is dissolved in blocking buffer. The label can be chosenfrom a variety of enzymes, including horseradish peroxidase (HRP),beta-galactosidase, alkaline phosphatase, glucose oxidase, etc.Sufficient time is allowed for specific binding to occur again, then thewell is washed again to remove unbound conjugate, and a suitablesubstrate for the enzyme is added. Color is allowed to develop and theoptical density of the contents of the well is determined visually orinstrumentally (measured at an appropriate wave length). The cutoff ODvalue may be defined as the mean OD+3 standard deviations (SDs) of atleast 50 serum samples collected from individuals from an area whereLyme disease is not endemic, or by other such conventional definitions.In the case of a very specific assay, OD+2 SD can be used as a cutoffvalue.

In one embodiment of an ELISA, a peptide or a mixture of peptides of theinvention is immobilized on a surface, such as a ninety-six-well ELISAplate or equivalent solid phase that is coated with streptavidin or anequivalent biotin-binding compound, such as avidin or nuetravidin, at anoptimal concentration in an alkaline coating buffer and incubated at 4°C. overnight. After a suitable number of washes with standard washingbuffers, an optimal concentration of a biotinylated form of a peptide orcomposition of the invention, dissolved in a conventional blockingbuffer, is applied to each well. A sample is then added, and the assayproceeds as above. Conditions for performing ELISA assays are well-knownin the art.

In another embodiment of an ELISA, a peptide or a mixture of peptides ofthe invention is immobilized on a surface, such as a ninety-six-wellELISA plate or equivalent solid phase via a fusion partner, e.g., BSA orMAPS. A sample is then added and the assay proceeds as above.

In another embodiment, the methods comprise an agglutination assay. Forexample, in certain embodiments, colloidal particles (e.g., colloidalgold, etc.) or latex beads are conjugated to peptides or compositions ofthe invention. Subsequently, the biological fluid is incubated with thebead/peptide conjugate, thereby forming a reaction mixture. The reactionmixture is then analyzed to determine the presence of the antibodies. Incertain embodiments, the agglutination assays comprise the use of asecond population of particles, such as colloidal particles (e.g.,colloidal gold, etc.) or latex beads, conjugated to (1) antibodiesspecific to the peptides of compositions of the invention, in the caseof a competition assay, or (2) antibodies capable of detecting sampleantibodies (e.g., anti-human IgG or IgM antibodies, anti-dog IgG or IgMantibodies, etc.), in the case of a sandwich assay. Suitableagglutination methods can comprise centrifugation as a means ofassessing the extent of agglutination.

In still other embodiment, peptide or compositions of the invention areelectro- or dot-blotted onto nitrocellulose paper. Subsequently, asample, such as a biological fluid (e.g., serum or plasma) is incubatedwith the blotted antigen, and antibody in the biological fluid isallowed to bind to the antigen(s). The bound antibody can then bedetected, e.g., by standard immunoenzymatic methods or by visualizationusing colloidal nanoparticles couples to secondary antibodies or otherantibody binding agents, such as protein A, protein G, protein L, orcombinations thereof.

It should be understood by one of skill in the art that any number ofconventional protein assay formats, particularly immunoassay formats,may be designed to utilize the isolated peptides of this invention forthe detection of Borrelia antibodies and infection by pathogenicBorrelia (e.g., B. burgdorferi) in a subject. This invention is thus notlimited by the selection of the particular assay format, and is believedto encompass assay formats that are known to those of skill in the art.

In certain embodiments, the sample used in the methods is a bodilyfluid, such as blood, serum, cerebral spinal fluid, urine, or saliva. Inother embodiments, the sample is a tissue (e.g., a tissue homogenate) ora cell lysate. In certain embodiments, the sample is from a wild animal(e.g., a deer or rodent, such as a mouse, chipmunk, squirrel, etc.). Inother embodiments, the sample is from a lab animal (e.g., a mouse, rat,guinea pig, rabbit, monkey, primate, etc.). In other embodiments, thesample is from a domesticated or feral animal (e.g., a dog, a cat, ahorse). In still other embodiments, the sample is from a human.

Much of the preceding discussion is directed to the detection ofantibodies against pathogenic Borrelia. However, it is to be understoodthat the discussion also applies to the detection of primed T-cells,either in vitro or in vivo.

It is expected that a cell-mediated immune response (e.g., a T-helperresponse) is generated, since IgG is produced. It is therefore expectedthat it will be possible to determine the immunological reactivitybetween primed T-cells and a peptide of the invention. In vitro this canbe done by incubating T-cells isolated from the subject with a peptideof the invention and measuring the immunoreactivity, e.g., by measuringsubsequent T-cell proliferation or by measuring release of cytokinesfrom the T-cells, such as IFN-γ. These methods are well-known in theart.

When a method of the invention is carried out in vivo, any of a varietyof conventional assays can be used. For example, one can perform anassay in the form of a skin test, e.g., by intradermally injecting, inthe subject, a peptide of the invention. A positive skin reaction at thelocation of injection indicates that the subject has been exposed to andinfected with a pathogenic Borrelia capable of causing Lyme disease, anda negative skin response at the location of injection indicates that thesubject has not been so exposed/infected. This or other in vivo testsrely on the detection of a T-cell response in the subject.

In another aspect, the invention provides methods of diagnosing Lymedisease in a subject. The subject can be a subject suspected of havingantibody against a causative agent of Lyme disease. The diagnosticmethod is useful for diagnosing subjects exhibiting the clinicalsymptoms of Lyme disease.

In certain embodiments, the methods comprise contacting a sample fromthe subject with a peptide of the invention, and detecting formation ofan antibody-peptide complex comprising said peptide, wherein formationof said complex is indicative of the subject having Lyme disease. Incertain embodiments, the methods comprise contacting the sample with amixture of two, three, four, or more (e.g., 5, 6, 7, 8, 9, 10, 15, 20,25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400, 500, ormore) different peptides of the invention. In certain embodiments, themethods comprise contacting the sample with a mixture of one or morepeptides or the invention and one or more other peptides (e.g., aBorrelia peptide, or antigenic fragment or epitope thereof, such as anOspA, OspB, DbpA, flagella-associated proteins FlaA (p37) and FlaB(p41), OspC (25 kd), BBK32, BmpA (p39), p21, p39, p66, p83, or VlsEprotein).

In certain embodiments, the peptide or each peptide in the mixture is anisolated (e.g., synthetic and/or purified) peptide. In certainembodiments, the peptide or mixture of different peptides is attached toor immobilized upon a substrate (e.g., a solid or semi-solid support).For example, in certain embodiments, the substrate is a bead (e.g., acolloidal or other type of particle or nanoparticle), a flow path in alateral flow immunoassay device (e.g., a porous membrane), a blot (e.g.,a Western blot, dot blot, or slot blot), a flow path in an analyticalrotor, or a tube or a well (e.g., in a plate suitable for an ELISAassay).

There are a number of different conventional assays for detectingformation of an antibody-peptide complex comprising a peptide of theinvention. For example, the detecting step can comprise performing anELISA assay, performing a lateral flow immunoassay, performing anagglutination assay, analyzing the sample using a Western blot, a slotblot, or a dot blot, analyzing the sample in an analytical rotor, oranalyzing the sample with an electrochemical, optical, oropto-electronic sensor. These different assays are described aboveand/or are well-known to those skilled in the art.

In certain embodiments, the sample is a bodily fluid, such as blood,serum, cerebral spinal fluid, urine, or saliva. In other embodiments,the sample is a tissue (e.g., a tissue homogenate) or a cell lysate. Incertain embodiments, the subject is a wild animal (e.g., a deer orrodent, such as a mouse, chipmunk, squirrel, etc.). In otherembodiments, the subject is a lab animal (e.g., a mouse, rat, guineapig, rabbit, monkey, primate, etc.). In other embodiments, the subjectis a domesticated or feral animal (e.g., a dog, a cat, a horse). Instill other embodiments, the subject is a human.

Kits

In yet another aspect, the invention provides kits. In certainembodiments, the kits comprise a peptide of the invention. In certainembodiments, the kits comprise two, three, four, or more differentpeptides of the invention. The peptides can comprise a sequence of SEQID NO: 1 or SEQ ID NO: 2. In certain embodiments, the peptides areattached to or immobilized on a solid support. For example, in certainembodiments, the solid support is a bead (e.g., a colloidal particle ora nanoparticle), a flow path in a lateral flow immunoassay device, aflow path in an analytical rotor, or a tube or a well (e.g., in aplate).

Reagents for particular types of assays can also be provided in kits ofthe invention. Thus, the kits can include a population of beads (e.g.,suitable for an agglutination assay or a lateral flow assay), or a plate(e.g., a plate suitable for an ELISA assay). In other embodiments, thekits comprise a device, such as a lateral flow immunoassay device, ananalytical rotor, a Western blot, a dot blot, a slot blot, or anelectrochemical, optical, or opto-electronic sensor. The population ofbeads, the plate, and the devices are useful for performing animmunoassay. For example, they can be useful for detecting formation ofan antibody-peptide complex comprising an antibody from a sample and apeptide of the invention. In certain embodiments, a peptide, a mixtureof different peptides of the invention, or a peptide composition of theinvention is attached to or immobilized on the beads, the plate, or thedevice.

In addition, the kits can include various diluents and buffers, labeledconjugates or other agents for the detection of specifically boundantigens or antibodies, and other signal-generating reagents, such asenzyme substrates, cofactors and chromogens. Other components of a kitcan easily be determined by one of skill in the art. Such components mayinclude coating reagents, polyclonal or monoclonal capture antibodiesspecific for a peptide of the invention, or a cocktail of two or more ofthe antibodies, purified or semi-purified extracts of these antigens asstandards, monoclonal antibody detector antibodies, an anti-mouse,anti-dog, anti-chicken, or anti-human antibody with indicator moleculeconjugated thereto, indicator charts for colorimetric comparisons,disposable gloves, decontamination instructions, applicator sticks orcontainers, a sample preparatory cup, etc. In one embodiment, a kitcomprises buffers or other reagents appropriate for constituting areaction medium allowing the formation of a peptide-antibody complex.

Such kits provide a convenient, efficient way for a clinical laboratoryto diagnose infection by a pathogenic Borrelia, such as a B.burgdorferi. Thus, in certain embodiments, the kits further comprise aninstruction. For example, in certain embodiments, the kits comprise aninstruction indicating how to use a peptide of the invention to detectan antibody to a Borrelia antigen or to diagnose Lyme disease. Incertain embodiments, the kits comprise an instruction indicating how touse a population of beads, a plate, or a device (e.g., comprising apeptide or a mixture of different peptides of the invention) to detectan antibody to a Borrelia antigen or to diagnose Lyme disease.

The peptides, compositions and devices comprising the peptides, kits andmethods of the invention offer a number of advantages. For example, theyallow for simple, inexpensive, rapid, sensitive and accurate detectionof Lyme disease, and avoid serologic cross-reactivity with otherconditions with “Lyme-like” symptoms, such as myalgias, arthralgias,malaise or fever, including conditions such as syphilis, chronicarthritis, and multiple sclerosis. This allows for an accuratediagnosis. Furthermore, a diagnostic test of the invention (e.g., anELISA assay, lateral flow immunoassay, or agglutination assay) is usefulin serum samples that contain anti-OspA antibodies or other antibodiesproduced in response to a vaccine based on the outer surface proteins ofBorrelia. A VlsE IR6 peptide of the invention does not cross-react withsuch antibodies, thereby allowing the differentiation of vaccinatedindividuals from individuals who were naturally infected with B.burgdorferi.

To the extent that any definitions in documents incorporated byreference are inconsistent with the definitions provided herein, thedefinitions provided herein are controlling. Although the invention hasbeen described with reference to the presently preferred embodiments, itshould be understood that various changes and modifications, as would beobvious to one skilled in the art, can be made without departing fromthe spirit of the invention. Accordingly, the invention is limited onlyby the following claims.

The disclosures, including the claims, figures and/or drawings, of eachand every patent, patent application, and publication cited herein arehereby incorporated herein by reference in their entireties.

What is claimed:
 1. A mixture of isolated peptides comprising three ormore different isolated peptides, wherein each isolated peptidecomprises (i) a sequence according toL-K-K-D-D-N-I-A-A-A-X₁₁-V-L-R-G-X₁₆-X₁₇-K-D-G-X₂₁-F-A-X₂₄-X₂₅ (SEQ IDNO: 1) wherein X₁₁ is an amino acid selected from the group consistingof V and L, X₁₆ is an amino acid selected from the group consisting of Land I, X₁₇ is an amino acid selected from the group consisting of A andV, X₂₁ is an amino acid selected from the group consisting of R, D andN, X₂₄ is an amino acid selected from the group consisting of I, W, andY, and X₂₅ is an amino acid selected from the group consisting of K andR; (ii) an N-terminal peptide sequence wherein the sequence isn₁-n₂-S-P-n₅-n₆-P (SEQ ID NO: 149) or a fragment thereof, wherein n₁ isan amino acid selected from the group consisting of A and V, n₂ is anamino acid selected from the group consisting of E and D, n₅ is an aminoacid selected from the group consisting of K and R, and n₆ is an aminoacid selected from the group consisting of K and R; and (iii) aC-terminal peptide sequence wherein the sequence is (a)V-c₂-E-G-c₅-Q-Q-E-G-A-Q-Q-P-S-C (SEQ ID NO: 150) or a fragment thereof,wherein c₂ is an amino acid selected from the group consisting of Q andR, and c₅ is an amino acid selected from the group consisting of V andA; or (b) A-V-c₃-E-G-c₆-Q-Q-E-G-A-Q-Q-P-S (SEQ ID NO: 151) or a fragmentthereof, wherein C3 is an amino acid selected from the group consistingof Q and R, and C6 is an amino acid selected from the group consistingof V and A.
 2. The mixture of claim 1, wherein each isolated peptide isconjugated to a ligand or wherein one or more of the isolated peptidesis biotinylated.
 3. The mixture of claim 1, wherein one or more of theisolated peptides is conjugated to: (i) streptavidin, polymericstreptavidin, avidin, or neutravidin; (ii) a carrier protein selectedfrom serum albumin, keyhole limpet hemocyanin (KLH), and animmunoglobulin Fc domain; (iii) a dendrimer; (iv) part of a multipleantigenic peptides system (MAPS); or (v) a colloidal particle,nanoparticle, colloidal nanoparticle, core-shell structure particle, orhollow nanosphere.
 4. The mixture of claim 1, wherein each isolatedpeptide is immobilized to a solid support.
 5. The mixture of claim 4,wherein each isolated peptide is immobilized to the solid supportthrough a terminal amino acid linking residue.
 6. The mixture of claim4, wherein the solid support is a bead, a flow path in a lateral flowimmunoassay device, a well in a microtiter plate, or a flow path in arotor.
 7. A method for diagnosing Lyme disease in a subject, the methodcomprising: contacting a sample from the subject with the mixture ofisolated peptides claim 1; and detecting formation of anantibody-peptide complex comprising one or more isolated peptides in themixture, wherein formation of the complex is indicative of the subjecthaving Lyme disease.
 8. The method of claim 7 wherein said detectingstep comprises (i) performing an ELISA assay, (ii) running a lateralflow assay, (iii) performing an agglutination assay, (iv) performing aWestern blot, a slot blot, or dot blot, or (v) running the samplethrough an analytical rotor.
 9. The method of claim 7, wherein saidsample is a bodily fluid.
 10. The method of claim 7, wherein said sampleis from a human, canine, or equine subject.
 11. A kit comprising themixture of isolated peptides of claim 1 and a labeling reagent capableof binding to an antibody that recognizes an epitope of one or moreisolated peptides in the mixture.
 12. The kit of claim 11, wherein thelabeling reagent is: (i) an anti-human, anti-canine, or anti-equineIgG/IgM antibody conjugated to a detectable label; or (ii) protein Aand/or protein G conjugated to a detectable label.
 13. The kit of claim12, wherein the detectable label is colloidal gold particles.